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Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences

  • Mikael V. Garabedian
    Mikael V. Garabedian
    Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
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  • , 
  • Zhihui Su
    Zhihui Su
    Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    More by Zhihui Su
    Orcidhttps://orcid.org/0000-0003-0239-5280
  • , 
  • Jorge Dabdoub
    Jorge Dabdoub
    Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
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  • , 
  • Michelle Tong
    Michelle Tong
    Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
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  • , 
  • Alexander Deiters
    Alexander Deiters
    Department of Chemistry, University of Pittsburgh, Philadelphia, Pennsylvania 15260, United States
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    Orcidhttps://orcid.org/0000-0003-0234-9209
  • , 
  • Daniel A. Hammer
    Daniel A. Hammer
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
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    Orcidhttps://orcid.org/0000-0002-3522-3154
  • , and 
  • Matthew C. Good*
    Matthew C. Good
    Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    *Email: [email protected]
    More by Matthew C. Good
    Orcidhttps://orcid.org/0000-0002-6367-1034
Cite this: Biochemistry 2022, 61, 22, 2470–2481
Publication Date (Web):August 2, 2022
https://doi.org/10.1021/acs.biochem.2c00250

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid–liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

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SPECIAL ISSUE

This article is part of the Protein Condensates special issue.

Condensation of proteins and RNAs into micron size “membraneless organelles” is a ubiquitous feature of all cell types facilitating subcellular compartmentalization in the absence of a lipid bilayer. (1,2) Polymers can demix from solution via liquid–liquid phase separation (LLPS), forming condensates via multiple weak interactions, often spreading across the polypeptide chain of intrinsically disordered proteins (IDPs). (3,4) Many biomolecular condensates are dynamic and exhibit liquid-like behavior including fission and fusion of individual condensates as well as rapid recovery from photobleaching both in vitro and in live cells. (5−8) These proteinaceous subcellular compartments serve a wide array of functions, concentrating reactants to accelerate reaction rates or sequestering client factors to shut down pathway activity. (1,9) Over the past decade, a number of activities regulated by biomolecular condensates have come into focus. These include those involved in ribosomal biogenesis (nucleoli), RNA processing (Cajal bodies), transcriptional regulation (enhancers and super enhancers), early development (P bodies), and stress response (stress granules). (5,10−17) Due to their diverse functionality and feasibility of forming compartments from a single type of disordered polypeptide, condensates are an attractive target for protein engineering, synthetic biology, and construction of new biologically inspired materials.
Unlike typical interactions in a protein complex among folded proteins, disordered polypeptides can self-assemble via multiple, relatively weak interactions between amino acids in the protein chain. (18) Because individual interactions are weak, valency is a fundamental strategy of increasing the propensity for LLPS. (19−21) If a short peptide sequence is repeated in a larger multimer, the longer polymer has a higher probability of homotypic and heterotypic interactions, thus lowering the required free energy for phase separation. (19,22−24) In short, the multivalency of side chain interactions profoundly effects the saturation concentration (Csat) for protein condensation, the liquid versus gel-like properties of condensates, and the kinetics of coarsening. (25−28) The extent of multivalency can be hard coded in the amino acid sequence or through post-translational modifications. (20,23,29−33) Both strategies have been explored to fine tune the extent of protein condensation under physiological conditions. (20,25,31,34−36) In cells, the viscoelastic properties of condensates can be altered by the presence of other multivalent polymers such as RNA. (37,38) For example, the C. elegans P granule protein LAF-1 is capable of LLPS independent of RNA in vitro, but the presence of RNA helps fluidize LAF-1 condensates. (6) In other cases, multivalency was used to lower the concentration of polypeptide required for self-assembly of elastin and resilin-like polymers. (39−41)
A challenge in protein engineering is how to regulate the valency of sequence-defined polymers in real time using genetically encoded components under physiological conditions. Simply encoding a longer, higher repeat polypeptide sequence removes the flexibility to alter polymer valency and for temporal control of phase separation. Several strategies exist to increase the chain length of existing polymers. One strategy uses chemical cross-linking such as intermolecular chain cross-linking via cysteine disulfide bonds. (42) Such a strategy was used to condense oleosin in a redox-responsive manner. (42) Short, elastin-like polypeptides can assemble into multivalent chains via cross-linking of non-native amino acids to control gelation. (43) However, due to their length, these sequences are not compatible with tagging with larger folded helices or domains, which in the case of IDRs would increase Csat to ranges of expression that cannot be achieved in cells. Native chemical ligation strategies can be used to multimerize polypeptides, although the chemical conditions may not be ideal for living systems. (44) For example, the Spycatcher–spytag system results in a covalent and irreversible conjugation of the two target peptides. (45) Alternatively, noncovalent interactions offer an attractive strategy to rapidly multimerize polypeptides using protein interaction domains. (46−48) Examples include coiled coils, small-molecule-induced dimerization of FRB-FKBP, optogenetic dimerization of DHFR, Halo, and optogenetic dimerization or multimerization domains. (34,47,49) Given the established utility of LAF-1 RGG IDR for engineering synthetic condensates (6,20,29,50,51) and the well-characterized biophysical features of its condensates, we chose to use this sequence for testing multimerization strategies in the present study.
The structure, affinity, and design of helical coiled-coil interaction domains are well characterized, inspiring a route for noncovalent control of IDR multivalency compatible via genetic encoding in living cells. Characterized by heptad sequence repeats, alpha-helical coils are common in endogenous proteins, and the rules of their interactions are well established for protein–protein binding. (52) They have been engineered to generate orthogonal synthetic heterodimeric helical peptides that have low nanomolar affinities and high sequence selectivity. (53−55) A growing body of literature has implemented helical coiled coils to create new connectivities and localizations in cells to regulate cellular functions. We previously used coiled-coil motifs, attached to an IDR and client proteins, to recruit clients to preformed IDR coacervates in vitro and later to insulate endogenous client enzymes within synthetic condensates to control the cell behavior. (20,29) Although heterotypic coiled coils have been successful in promoting scaffold–client interactions, their utility for stitching together disordered polypeptides to control protein phase separation has not been explored. Two well-validated examples that we focus on in this work include SYNZIPs and Parallel Peptide Pairs. (55−57) In both cases, a variety of short, parallel coiled-coil pairs have been constructed and shown to be orthogonal, interacting with only its designated, cognate coil and no others in the set. (56,57) As such, these tools and others like them are ideal for tagging exogenously or endogenously expressed proteins without disrupting their functionality to promote specific, noncovalent binding or dimerization. (29)
Additional strategies for inducible multimerization or clustering of IDRs leveraged light-responsive protein interaction domains, which have been instrumental in studying the LLPS activity of a number of disordered sequences in cells under illumination conditions. (50,58,59) Such tools have been used to enhance or control biochemical reactions in cell culture using the presence and extent of illumination. (60,61) Whereas optogenetics require sustained illumination and may be challenging to implement biochemically in vitro, chemogenic and optochemical approaches utilizing small molecular dimerizers and Halo-DHFR or FRB-FKBP domains have been used both in vitro and in cells. (49,62) For example, anchoring native factors away from substrates either by relocalization to other native organelles or by insulation within synthetic condensates has been shown to be successful in controlling the cell polarity and proliferation in a predicable manner. (29,63,64)
In this work, we characterized sets of short, heterodimeric coiled-coil modules to promote the multivalency of a model IDR, the Arg/Gly-rich RGG domain of C. elegans P granule protein LAF-1 and regulate protein condensation in vitro and in live cells. We chose the LAF-1 RGG IDR which has been used to engineer synthetic condensates and whose biophysical determinants for phase separation are understood. (6,20,29,50,51) We first identified optimal strategies for tagging the RGG IDP with coiled coils to promote dimerization and condensate formation in vitro. To achieve temporal chemogenic control over condensation, we constructed RGG domains fused to FRB and FKBP domains in vitro and in cells. We also show optical control of condensate formation using a photocaged dimerizer within cell-like compartments in vitro and in live cells, a strategy that requires only a short pulse of light to stably form condensed phases. Collectively, this study reveals new strategies for real-time control of IDP valency providing a regulatory toolkit for protein condensate assembly with applications in protocell engineering and synthetic biology.

Materials and Methods

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Protein Expression and Purification

Plasmids encoding N-terminally 6xHis-tagged RGG constructs with coiled-coil tags and 6xHis mCherry-FRB were transformed into BL21(DE3) E. coli cells (Thermo Fisher Scientific; Waltham, MA). Cultures were grown in Luria Broth (LB) containing kanamycin at 37 °C to an OD600 of 0.6–0.8, and expression was induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 °C overnight. Cell pellets were collected and stored at −80 °C.

RGG-FKBP and RGG-FRB Purification

Bacterial cell pellets were thawed, resuspended in lysis buffer (50 mM HEPES, pH 6.8, 1 M NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol) containing complete EDTA-free protease inhibitor cocktail (Roche; Mannheim, Germany), and lysed for a total of 3 min of sonication at 50% power using a Branson Sonifier. Lysates were clarified by centrifugation at 13 000 rpm (20,064g) for 20 min in an F21S-8x50y rotor (Thermo Fisher Scientific) at 37 °C and incubated with 0.5 mL of Ni–NTA beads (Thermo Fisher Scientific) at room temperature for 1 h. Beads were then washed three times with 10 mL of lysis buffer. Proteins were eluted by addition of lysis buffer containing 500 mM imidazole and 1 mM DTT. Elutions were diluted to 3 mg/mL in lysis buffer containing 1 mM DTT and dialyzed overnight into single RGG storage buffer (500 mM NaCl, 20 mM HEPES, pH 6.8, 1 mM DTT) using 10 kDa cutoff Slide-A-Lyzer membrane cassettes (Thermo Fisher Scientific). Proteins were concentrated by centrifugation in 4 mL of Amicon filter concentrators with a 10 kDa cutoff (Millipore Sigma; Burlington, MA). TCEP (1 mM) was added prior to snap freezing and storage at −80 °C

Purification of Tandem, RGG-RGG, Fluorescent Tracer (RGG-GFP-RGG), 6xHis-RGG Domains Tagged with Coiled-Coil Dimerizers (P3-6, SZ1/2), and 6xHis-mCherry-FRB

Pellets were thawed and resuspended in lysis buffer. RGG polypeptides that included P3-6 or tags and mCherry-FRB were resuspended in lysis buffer containing 50 mM Tris-HCl, pH 8.5, 1 M NaCl, 20 mM Imidazole, 1 mM β-mercaptoethanol, and a dissolved tablet of protease inhibitor cocktail (Roche). RGG constructs with SZ1/2 tags were treated similarly, but lysis buffer contained 20 mM HEPES pH 6.8. Cells were lysed, cleared by centrifugation, and incubated with Ni–NTA beads as above. Beads were then washed in three column volumes of lysis buffer and eluted with lysis buffer containing 500 mM imidazole and 1 mM DTT. Elutions were diluted to 3 mg/mL in lysis buffer containing 1 mM DTT as above and dialyzed overnight into storage buffer. Constructs with P3–P6 and mCherry-FRB: 1 M NaCl, 20 mM Tris-HCl, pH 8.5, 1 mM DTT. Constructs with SZ1 and SZ2: 1 M NaCl, 20 mM Tris-HCl, pH 6.8, 1 mM DTT. Proteins were concentrated by centrifugation in Amicon filter concentrators with a 10 kDa cutoff before addition of 1 mM TCEP and storage at −80 °C. In all cases, protein concentrations were determined by A280 and Bradford assay (BioRad).

Turbidity Measurements

Protein aliquots were thawed and solubilized at 50 °C and then diluted to 6 μM in buffer to adjust to a final salt concentration of 150 mM in 20 mM, Tris-HCl, pH 8.5. SYNZIP-tagged constructs were similarly adjusted to 10 μM in 150 mM NaCl, 20 mM HEPES, pH 6.8. The protein mixture, 60 μL in volume, was added to quartz microcuvettes (10 mm path length) (Starna Cells, Inc. Atascadero, CA). Cuvettes were inserted into a Cary 3500 UV–vis spectrophotometer controlled by an Agilent multizone Peltier temperature controller (Agilent Technologies; Santa Clara, CA). To test kinetics of rapamycin-induced dimerization and phase separation of FRB and FKBP-tagged RGG constructs, rapamycin (Sigma-Aldrich; St. Louis, MO) was spiked into the protein mixtures to a final concentration of 10 μM and absorbance at 600 nm was measured over time. For mapping the temperature-dependent phase separation, protein mixtures were applied to quartz cuvettes preincubated at 50 °C. Cuvettes were then inserted into the preheated spectrophotometer, set to 50 °C, and samples were cooled to 5 °C at a rate of 1 °C/min while measuring the absorbance at 600 nm.

Imaging of Protein Condensation In Vitro

Fluorescence microscopy imaging of protein droplet formation was performed at ambient temperature (approximately 22 °C) on an Olympus IX81 inverted confocal microscope (Olympus Life Science; Tokyo, Japan) equipped with a Yokogawa CSU-X1 spinning disk, mercury lamp, 488 and 561 nm laser launches, iLas-targeted laser system for photobleaching and an iXon3 EMCCD camera (Andor; Belfast, UK). Multidimensional acquisition was controlled by MetaMorph software (Molecular Devices; Downingtown, PA). Samples were illuminated using a 488 nm laser and imaged through a 100×/1.4 NA oil-immersion objective. To image in vitro droplet formation, proteins were thawed at 50 °C, diluted to 4 μM in a buffer containing 150 mM NaCl and 20 mM Tris-HCl (pH 8.5 unless otherwise specified), and placed on custom-fabricated acrylic gasket chambers adhered to glass coverslips.

Chemical Dimerization

The fluorescent tracer, RGG-GFP-RGG, was present in the protein mixture at 0.1–0.2 μM to track condensation in the 488 nm channel. Chamber wells had been previously passivated overnight in a solution of 10 mg/mL BSA (Thermo Fisher Scientific) at room temperature and then rinsed with sterile ddH2O immediately prior to use. Condensate formation of FRB and FKBP-tagged proteins was induced by addition of rapamycin (Sigma-Aldrich) at a final concentration of 10 μM per reaction. Tandem RGG constructs in the same buffer conditions noted above formed condensates in the absence of additional components. Condensate formation was monitored by time-lapse imaging with bright-field transmitted light and via 488 nm fluorescence. FRAP experiments were conducted on the same microscope using 405 nm light from an iLas targeted laser system. For photobleaching of internal regions of droplets, ROIs of similar sizes were selected and bleached. For photobleaching of whole droplets, a circular ROI encompassing an entire droplet was selected and photobleached as above.

Optical Uncaging of dRap for Light-Induced Condensation

Proteins mixtures were assembled in a dark room. Proteins were diluted to 10 μM in a reaction in a buffer containing 150 mM NaCl and 20 mM HEPES, pH 6.8, and supplemented with 5 μM dRap. Each molecule of dRap, upon uncaging, liberates two molecules of Rap. The protein mixture was then encapsulated inside cell-size water-in-oil emulsions by repeated pipetting of 1 μL of aqueous phase within 50 μL of a 5% (w/v) mixture of Cithrol DPHS (Croda, Inc. Edison, NJ) dissolved in mineral oil (Sigma-Aldrich). This emulsion mixture was then applied to wells in custom imaging chambers that had been pretreated overnight with mineral oil. Emulsions were allowed to settle for 20 min in the dark. To induce condensation, emulsions were subjected to 30 s total of continuous 405 nm light from a mercury lamp applied in steps through the Z planes. Droplet formation inside emulsions was then monitored via time-lapse microscopy. Occasionally, emulsions would drift, and the field of view was recentered manually between imaging intervals.

Yeast Procedures

Standard methodologies were followed for all experiments involving S. cerevisiae. (65,66) For Rap and dRap-mediated droplet assembly, a tor1–1 fpr1Δ::KANMX6 strain in the BY4741 genetic background was used. All other yeast strains were of the YEF473 genetic background. RGG constructs with a coiled coil or FRB and FKBP tags were cloned downstream of a galactose-inducible GAL1 promoter and integrated into the yeast URA3 locus using the Yiplac211 integrating vector or LEU2 locus using the Yiplac128 integrating vector by linearizing plasmid with EcoRV just before transformation. All yeast transformations were performed by the standard lithium acetate method.
To induce expression of RGG constructs in yeast, cells were first grown to saturation overnight in liquid YPD media in a 25 °C shaking incubator. Cells were then washed three times in sterile water, diluted in YP + 2% Raffinose, and incubated in a 25 °C shaking incubator for 6–8 h. Finally, yeast cells were diluted to an OD600 of 0.3 in YP + 2% galactose. Induction was allowed to proceed overnight in the same shaking incubator. The final OD600 of cultures used for experiments was between 0.4 and 0.8.

Image and Data Analysis

Quantitation of In Vitro Protein Condensate Formation

We used image segmentation in ImageJ. Two-dimensional maximum intensity projections in the 488 nm channel (RGG-GFP-RGG tracer) were generated for each time point and converted to 8-bit images. A binary mask was generated by automated thresholding with the Intermodes algorithm, objects were cleared from the boundary, and a watershed function was used to split objects. The particle analysis function in ImageJ was used to segment condensates. This provided the droplet number and areas from maximum intensity projections at each time point. Conversion of the droplet areas to volume was performed assuming a spherical shape.
FRAP experiments were analyzed by placing an appropriately sized ROI over the photobleached area of each droplet. The fluorescence profile over time for each ROI over time was recorded, and the maximum value prior to photobleaching was set to 1. Results from FRAP experiments for each type of droplet were then pooled, and the average recovery is shown with standard deviation.
Analysis of condensate formation in cells was performed in a similar manner in ImageJ. Time-lapse images were converted to maximum intensity projections. Individual objects (cells) were first cropped to facilitate condensate segmentation. Images were corrected for bleaching using an exponential fit. Condensate masks were generated from 8-bit images thresholded by the intermodes algorithm. In a small number of cases, despeckling was required prior to thresholding. Cells that could not be reliably thresholded were excluded from analysis. After generating a mask, particle analysis was performed in ImageJ as above to generate the object number and size. The number of droplets is reported as generated by this analysis, and volumes were calculated from the 2D areas assuming objects are spherical.

Results and Discussion

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Modular, short protein helical coiled-coil bundles have been used to dimerize and target components in cells for nanoscale origami of protein structures and to generate switches via protein engineering. (67−69) Previously, we demonstrated the feasibility of recruiting exogenous proteins to an IDP condensate by tagging scaffold and client proteins with cognate SYNZIP (SZ1 and SZ2) coiled coils. (29) We wondered whether we could also use these coiled coils to stitch together higher order assemblies of the model IDR from LAF-1, the RGG domain. We decided to test a handful of coiled coils including the 48 aa SYNZIPs (56) and 33 aa Parallel Peptide Pairs. (57) We cloned these coiled coils on the N or C terminus of the LAF-1 RGG domain and characterized the phase separation behavior of individual and paired sets of proteins using microscopy and spectrophotometric turbidity assays (Figure 1). The LAF-1 RGG domain displays the upper upper critical solution temperature (UCST) behavior; it is miscible at high temperatures and as the temperature is lowered will condense at its phase boundary or “cloud point”, resulting in turbid solutions. (70) Using microscopy, we could image condensate assembly at ambient temperature either in bright field or with fluorescence using a small amount of fluorescent RGG tracer (RGG-GFP-RGG) significantly below its Csat, such that it would not condense alone. As a validation of our biochemical conditions, we show that RGG-RGG (constitutive dimer) formed robust condensates that could be visualized by fluorescence microscopy. (Figure S1A). Under these same conditions, the single RGG domain will not condense because of its much higher Csat. (20)

Figure 1

Figure 1. Tuning Csat for condensation using noncovalent RGG multimerization via coiled-coil pairs. (A) Schematic of assembly of higher order RGG disordered polypeptides by genetic fusion to cognate pairs of helical coiled coils. In OFF (monomer) state, monomer concentrations should be below their Csat, and in ON (dimer) state, higher polymer valency (length) lowers Csat such that the dimer condenses into liquid-like droplets. (B, D, and F) Representative fluorescence microscopy images of condensates formed using 4 μM RGG monomer concentrations, 150 mM salt, pH 8.5. (B) images of SZ1-RGG, RGG-SZ2, and a mixture of SZ1-RGG and RGG-SZ2. (C) Turbidity measurements at A600 for 6 μM concentration of the control RGG-RGG dimer, indicated monomers, or mixtures of monomers, over a range of temperatures. (D) Images of P3-RGG, RGG-P4, and combination of P3-RGG and RGG-P4. (E) Turbidity measurement using a 6 μM concentration of the indicated control, monomer, and mixed monomer over a range of temperatures. (F) Representative images of RGG-P5, RGG-P6, and a mixture of RGG-P5 and RGG-P5. (G) Turbidity measurement using a 6 μM concentration of the indicated control dimer, monomer, and mixed monomer over a range of temperatures.

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Using these assay conditions, we set out to test whether tagged RGG remains miscible (OFF) in the monomer form but could condense (ON) as a mixed heterodimer. We first tested tagging a single RGG domain tagged with a folded SZ1 or SZ2 coiled coil under physiological conditions at 4 μM protein concentration. Somewhat unexpectedly, SZ1-RGG and RGG-SZ2 both formed condensates on their own (Figure 1B), suggesting a lower Csat than that of the untagged RGG alone. (20) By mixing this cognate pair we expected the RGG domain to dimerize and for condensates to grow larger. Indeed, we observe this behavior and a shift in critical temperature in the turbidity assay (Figure 1C). The lowered Csat of SYNZIP-tagged single RGG constructs may be explained by chain collapse due to reciprocal charges between the RGG domains (positively charged) and the coiled-coil tag (negatively charged). The RGG domain is enriched with positively charged arginine residues across its sequences, which are involved in π–π contacts and contribute to its phase separation. (51) Electrostatic interactions between the SYNZIPs and the RGG domain could result in partial chain collapse, and several studies have demonstrated that chain collapse is an important feature in the phase separation of disordered proteins; single-chain collapse theory has been used to predict phase boundaries of disordered proteins. (3,71,72) It is plausible that by introducing a motif with reciprocal charge, we promoted increased chain collapse, leading to a lower overall Csat and greater propensity to phase separate in the conditions used in the assay.
Because individual RGG domains tagged to SZ1 with SZ2 formed condensates on their own in the OFF state of our assay conditions, we chose to characterize additional coiled-coil pairs. LAF-1 RGG domains tagged with different parallel coiled coils (P3 or P4) at either terminus do not condense at physiological conditions at a 4 μM protein concentration (Figure 1D, Figure S1B). Yet, when paired, they form micron scale condensates within minutes of mixing protein solutions. By increasing the protein concentration, we identified the phase boundary for RGG-P4 alone, yet P3-RGG remained miscible, suggesting the monomers indeed have a higher Csat than the dimer (Figure S1B). By varying the position of the P3 and P4 tags on the RGG termini, we observed no coacervation of any of the monomer variants under these experimental conditions. Of the mixed pairs at the same molar concentrations, P3-RGG + RGG-P4, RGG-P3 + P4-RGG, and RGG-P3 + RGG-P4 displayed condensation (Figure S1C). Importantly, this condensation is specific to heterodimerization as mixtures of noncognate pairs of tagged RGG did not generate condensates (Figure S1D). The lowest critical concentration appeared to be for P3-RGG + RGG-P4 as it formed the largest condensates. The mixed pair of P3-RGG + P4-RGG did not form condensates, suggesting that N-terminal tagging may have an effect on either dimerization or RGG condensation. We note that RGG contains an important 10 aa patch, residues 21–30, which promote condensation. (51) Turbidity measurements on P3-RGG and RGG-P4 support the microscopy results and suggest that they are an excellent pair to control RGG valency and Csat (Figure 1E). We also tested the phase separation of RGG tagged with a different set of parallel coiled coils, P5 and P6 (Figure 1F and 1G). At a 4 μM protein concentration, pH 8.5, and physiological salt, the P5-RGG and RGG-P6 monomers showed no visible condensation in the OFF state (Figure 1F). The monomers also showed very little turbidity, even at lower temperatures, and appeared to heterodimerize nicely, creating a large increase in cloud point for the mixed pair, features that suggest they might be an ideal pair for controlling RGG valency. However, at pH 6.8, the same concentration of individual monomers condenses on their own (Figure S1E). Notably, the P3- and P4-tagged RGG monomers do not exhibit such pH sensitivity. Taken together, this data suggested that it is feasible to increase valency via protein interaction tags and that the type and position of the coiled coil influences the level of miscibility in the OFF (monomer) state and efficacy of condensates in the ON state (pair, heterodimer). In addition, these data suggest that the P3 and P4 heterodimers are quite compatible with dimerization and condensation of the LAF-1 RGG IDP and may be broadly useful for generating higher order disordered polymers.
Having validated LAF-1 RGG heterodimerization as a means to increase polymer valency and self-assembly into condensates, we sought to induce coacervation with temporal precision by adding a small molecule. We fused chemically responsive FRB and FKBP tags to the RGG domains (Figure 2). FRB and FKBP domains form a ternary complex with rapamycin (Rap), and thus, when linked to RGG domains, Rap induces formation of a divalent RGG polymer (Figure 2A). Because folded FRB and FKBP domains are larger than the coiled coils, they more significantly alter the order-to-disorder ratio of the polypeptide and increase Csat. As a result, a slightly higher protein concentration was needed to visualize condensation of the dimerized state. A covalently linked RGG-RGG control dimer robustly phase separates into liquid droplets at ambient temperature and 10 μM concentration, consistent with previous observations. Monomeric RGG domains fused to FRB and FKBP tags are miscible in the same aqueous solution (Figure 2B). Addition of Rap triggers rapid and robust phase separation to form micrometer-scale condensates that enrich with the RGG-GFP-RGG tracer (Figure 2B–D, Movie S1). To quantify the kinetics of the heterodimer assembly, we measured turbidity over time in spectrophotometric turbidity assays after addition of Rap (Figure 2E). Reactions lacking Rap do not become turbid, consistent with in vitro microscopy. Addition of Rap results in rapid solution turbidity with a t1/2 of ∼10 s (Figure 2E), demonstrating efficient system responsiveness resulting from multimerization of RGG domains. We also used turbidity assays to compare the UCST behavior of Rap-induced RGG dimers to our control RGG-RGG (Figure 2F). Monomeric RGG domains fused to FRB or FKBP in the absence of Rap have critical temperatures similar to single RGG domains, (20) and dimerization in the presence of Rap results in turbidity curves similar to the RGG-RGG control. Finally, we assayed the liquidity of these condensates using fluorescence recovery after photobleaching (FRAP) to measure the dynamics of a fluorescent tracer localized to the condensed phase. Fluorescence of the RGG-GFP-RGG tracer in control condensates and those formed by Rap-mediated dimers rapidly recovers after photobleaching and shows very similar recovery kinetics both via internal diffusion of fluorescent molecules as well as via diffusion from outside condensates (Figure 2G and 2H and Figures S2A and S2B). Taken together, these data demonstrate a strategy for chemogenic heterodimerization of IDP scaffolds to increase the valency and induce formation of liquid-like condensates.

Figure 2

Figure 2. Temporal control of IDR multimerization and phase separation in vitro. (A) Schematic representation for chemogenic dimerization of RGG polypeptides to form mesoscale liquid-like protein condensates. (B) Representative images of liquid droplet formation through increased domain valency upon addition of dimerizer, Rap. Recombinant RGG-FKBP and RGG-FRB proteins, in the absence of Rap, do not form condensates in a buffer containing 10 μM protein and 0.2 μM tracer (RGG-GFP-RGG). Scale bar, 10 μm. Addition of Rap to the reaction rapidly induces dimerization, causing condensate formation similar to the RGG-RGG constitutive dimer. (C and D) Quantitation of the kinetics of droplet formation upon equimolar addition of Rap. Average of three independent trials. Shaded area, StDev. (E) Kinetics of solution clouding after addition of dimerizer in spectrophotometric turbidity assays; average of three experiments area shown. (F) Phase transition temperature measured by turbidity assay shows induced dimerization of RGG-FKBP and RGG-FRB with Rap shifts the cloud point to higher temperatures, similar to constitutive RGG-RGG dimer; average of three experiments. (G) Representative images from photobleaching and recovery of condensates composed of Rap-mediated RGG-FKBP/RGG-FRB dimers marked by 0.2 μM RGG-GFP-RGG tracer. Scale bar 5 μm. (H) Quantification of FRAP indicating similar recovery kinetics of Rap-induced vs constitutive RGG-RGG dimers. n = 30 condensates from two independent trials.

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A useful feature of the FRB and FKBP domains is that their dimerization can also be optically regulated. (73) A challenge of optogenetic dimerization domains is that their association requires sustained illumination, and many of these systems are difficult to reconstitute in vitro. We sought to trigger irreversible condensation in vitro using only seconds of illumination. To achieve this, we utilized a photocaged version of rapamycin (dRap) (47,73) in which a cage occludes the FRB binding sites, thereby preventing FRB-FKBP dimerization in the dark state (Figure 3A and 3 B). To achieve sufficient levels of illumination and mimic a cellular context, we encapsulated RGG-FRB and RGG-FKBP proteins with dRap in cell-size water-in-oil emulsions. This approach confines the reaction to picoliter volumes which can be entirely illuminated on a microscope. Prior to illumination, we observed an evenly dispersed signal from our RGG-GFP-RGG tracer, indicating that no LLPS had occurred (Figure 3C). Upon exposure to 405 nm light, we observed rapid formation of protein droplets, visible within tens of seconds (Figure 3C and 3D, Movie S2). These data demonstrate a robust method for control of RGG domain valency in real time to drive stable protein condensation, responsive to both small-molecule and optical triggers.

Figure 3

Figure 3. Optochemical regulation of IDR condensation. (A) Chemical structure of photocaged rapamycin, dRap. (B) Schematic of approach: RGG domains fused to FKBP or FRB tags do not dimerize in the presence of dRap. Upon illumination, dRap is uncaged to Rap, resulting in dimerization of RGG-FKBP and RGG-FRB. (C) Pre- and postillumination images of water-in-oil emulsions, stabilized by Cithrol DPHS surfactant, encapsulating 10 μM each of RGG-FKBP and RGG-FRB, 0.2 μM RGG-GFP-RGG as a fluorescent tracer, and 5 μM dRap. Dotted line is the emulsion boundary. Prior to illumination, GFP signal is diffuse, indicating no condensation. After 30 s of illumination and uncaging of Rap, RGG condensates appear, indicating dRap uncaging and protein dimerization. Scale bar 10 μm. (D) Kinetics measured from images of optically induced droplet formation inside emulsions, as in C. n = 10 emulsions. Shaded area, StDev.

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To demonstrate that our inducible RGG dimerization and condensation system can be extended to living systems, we encoded the monomer scaffold in a model single-cell organism. Budding yeast, S. cerevisiae, is a well-established system for studying aggregate formation and LLPS in vivo (Figure 4). We genomically integrated sequences encoding single RGG domains fused to FRB and FKBP under the control of an inducible yeast GAL1 promoter. We included a GFP tag on one of the constructs (pGAL1-RGG-GFP-FKBP) in order to visualize condensate formation (Figure 4A). We induced expression of these single RGG constructs via galactose and observed only a diffuse GFP signal, indicating that without dimerizer they do not undergo LLPS (Figure 4B). Upon addition of Rap, fluorescent puncta appeared. Condensates could be initially observed within tens of seconds and formed micron-size structures on the order of minutes (Movie S3), increasing in both number and size over time (Figure 4C and Figure S3A). We then tested whether we could induce the assembly of stable membraneless organelles using a single, short period of light exposure. Using the same strain and encoded constructs as above, we added the photocaged dRap to the cells in media. Upon a 10 s pulse of 405 nm light, round puncta appeared and grew in number and size over time (Figure 4D and 4E), Rap addition or dRap optical uncaging, condensates also grew in size (Figure 4E and Figure S3B). Further, the kinetics of droplet formation is very similar in both strategies above (Figure S3C). In contrast to the available optical LLPS systems in vitro, this strategy requires only a single short pulse of light to induce dimerization of a disordered sequence and cause protein coacervation.

Figure 4

Figure 4. Leveraging induced dimerization to trigger synthetic condensate formation in living cells. (A) Scheme for encoding and expression of RGG polypeptides in cells, sensitive to small-molecule-induced dimerization and condensation. RGG-GFP-FKBP and RGG-FRB constructs are integrated into the yeast genome controlled by an inducible GAL1 promoter. (B) Representative images for strains described in A, showing addition of 20 μM Rap triggers condensate formation within minutes in live cells. (C) Average number of droplets formed per yeast cell in the n = 105 cells. Shaded area, 95% CI. (D) Optical regulation of condensation in cells following illumination to photouncage dRap. (E) Average number of droplets formed per cell following 10 s of 405 nm laser illumination (n = 66 cells). Shaded area, 95% CI.

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We also wanted to test whether chemogenic dimerization could be utilized to control recruitment of clients or cargo to the condensed phase, mimicking enzyme partitioning to membraneless organelles. To test the specificity and temporal kinetics of client recruitment, we used a model cargo, mCherry-FRB, and imaged its enrichment in condensates formed from either RGG-RGG or heterodimers of RGG-FKBP + RGG-FRB in the presence of Rap (Figure 5A and 5B). The cargo does not enrich in control RGG-RGG condensates, whereas it is recruited to and enriched in condensates of dimerized RGG-FKBP + RGG-FRB (Figure 5C). This suggests selective client recruitment to the condensed phase dependent on dimerizing to RGG-FKBP. Overall, these data demonstrate a proof-of-concept for rapid and stable multimerization to form condensates both in vitro and in cell culture and provide new avenues for temporally regulating condensates assembly and composition, useful features for cellular engineering.

Figure 5

Figure 5. Selective cargo recruitment to condensates via chemogenic dimerization. (A) Schematic of RGG-RGG condensates, which cannot selectively recruit FRB-tagged client protein (mCherry-FRB). Client does not enrich in condensates because it cannot interact with free RGG polypeptides. (Right) Representative fluorescent images of condensates from 10 μM RGG-RGG marked with 0.2 μM RGG-GFP-RGG tracer and 5 μM of client mCherry-FRB. Client is excluded from RGG-RGG condensates. (B) Scheme for cargo recruitment via rapamycin through dimerization with RGG-FKBP which partitions to condensed phase. (Right) Representative images of condensates from 10 μM each of RGG-FKBP and RGG-FRB in the presence of 10 μM Rap and 0.2 μM RGG-GFP-RGG tracer. Addition of 5 μM mCherry-FRB (client) results in robust and selective enrichment to condensed phase. (C) Kinetics of client enrichment after addition of mCherry FRB as in A and B from three independent experiments. Shaded area, 95% CI.

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Conclusions

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Characterizing the behavior of disordered protein sequences that self-assemble into condensed phases is important for understanding the biology of membraneless organelles and in bioengineering to generate gel-like and other materials for a range of therapeutic and industrial applications. (40,61,74−76) Similar to previous work with multivalent folded domains, (36,77) we demonstrated that the valency of an IDR determines critical concentrations for phase separation in vitro. (20) Although proteolytic cleavage has been used to reduce IDR valency, (20,78) we were interested in the converse, building up multivalency of an IDR sequence to regulate protein condensation in a predictable manner. Here, we systematically engineered noncovalent dimerization of the LAF-1 RGG domain via coiled-coil motifs and further demonstrated real-time control of IDR condensation and client recruitment using folded optochemical dimerization domains. On the basis of our initial findings, one can also imagine increasing valency to trimeric and tetramer IDRs to alter Csat or promote temperature-resistant LLPS in cells and also to fine tune the desired physicochemical properties of the condensed phase.
We note there may be several reasons why the terminal position of the coiled coil can influence the LLPS of the monomer IDR. First, the LAF-1 RGG domain harbors a well-conserved and critical motif at its N-terminal end, aa 21–30; (51) and thus, tagging may interfere with clustering of this motif. Also, the RGG domain has well-dispersed charge along the polypeptide chain, but it also has slightly more positive charge at its C-terminal end, which may interact with the net negative charge of the helical coils whose isoelectric points are between 4 and 5 and thus affect phase separation by altering the kinetics of chain collapse. Future study using positively charged coiled coils would be of interest. Despite the size and structured nature of FKBP and FRB tags, these tagged constructs formed condensates that behaved similarly to dimeric RGG controls, suggesting that tagging with folded domains did not significantly alter condensation behavior. We note that this is likely due to use of a 168 aa IDR, and the ratio of IDR to folded protein molecular mass can certainly affect LLPS.
Multivalency has been used as a useful strategy for driving self-assembly and coacervation for both structured and disordered proteins. (20,23,33) Our study provides paradigms for sequential multimerization of IDRs for applications in materials science, synthetic biology, and cellular engineering. The short coiled coils are readily knocked in using CRISPR to native gene loci without disrupting endogenous protein function. (29) Further, more complex assemblies using multiple sets of coiled coils in protein origami could be used to build novel architectures that potentially nucleate condensation.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.2c00250.

  • Additional experiments including varying concentrations and pH of mixtures of RGG tagged with coiled coils, FRAP experiments, quantification of yeast data, and legends describing supplemental movies (PDF)

  • Bright-field and 488 nm channel images of condensate formation from 10 μM RGGFKBP and RGG-FRB (AVI)

  • Condensate formation in cell-sized emulsions (AVI)

  • Rapamycin induced condensate formation in live yeast cells (AVI)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
    • Matthew C. Good - Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States;  Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States;  Orcidhttps://orcid.org/0000-0002-6367-1034;  Email: [email protected]
  • Authors
    • Mikael V. Garabedian - Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    • Zhihui Su - Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States;  Orcidhttps://orcid.org/0000-0003-0239-5280
    • Jorge Dabdoub - Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    • Michelle Tong - Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
    • Alexander Deiters - Department of Chemistry, University of Pittsburgh, Philadelphia, Pennsylvania 15260, United States;  Orcidhttps://orcid.org/0000-0003-0234-9209
    • Daniel A. Hammer - Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States;  Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States;  Orcidhttps://orcid.org/0000-0002-3522-3154
  • Author Contributions

    M.V.G., Z.S., D.A.H., and M.C.G. conceptualized the project and designed experiments. A.D. synthesized the small molecules. M.V.G., Z.S., J.D., and M.T. performed the experiments. M.V.G. and Z.S. analyzed data. M.V.G., Z.S., D.A.H., and M.C.G. wrote the manuscript.

    Author Contributions

    M.V.G. and Z.S.: These authors contributed equally to this work.

  • Funding

    This study was supported by a National Institute of Biomedical Imaging and Bioengineering R01 grant, EB028320 (M.C.G. and D.A.H.). Biochemical characterization of disordered proteins was partly funded by National Science Foundation (NSF) MRSEC Seed grant DMR1720530 (M.C.G. and D.A.H.). Synthesis of optochemical dimerizers was supported by NSF grant CHE-1404836 (A.D.). Conceptual development of IDR multimerization and investigator salary support was supported in part by Department of Energy BES Biomolecular Materials grant DE-SC0007063 (M.C.G. and D.A.H.).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Andrea Stout and the Penn CDB Microscopy Core for imaging and support as well as Erfei Bi and Anuj Kumar for yeast strains and technical expertise for yeast work.

References

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    The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent rRNA. A no. of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying mol. interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here the authors utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. The authors show that above a threshold concn. of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal mol. mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. The authors propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.
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  11. 11
    Feric, M.; Vaidya, N.; Harmon, T. S.; Mitrea, D. M.; Zhu, L.; Richardson, T. M.; Kriwacki, R. W.; Pappu, R. V.; Brangwynne, C. P. Coexisting Liquid Phases Underlie Nucleolar Subcompartments. Cell 2016, 165 (7), 1686– 1697,  DOI: 10.1016/j.cell.2016.04.047
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    11
    Coexisting liquid phases underlie nucleolar subcompartments
    Feric, Marina; Vaidya, Nilesh; Harmon, Tyler S.; Mitrea, Diana M.; Zhu, Lian; Richardson, Tiffany M.; Kriwacki, Richard W.; Pappu, Rohit V.; Brangwynne, Clifford P.
    Cell (Cambridge, MA, United States) (2016), 165 (7), 1686-1697CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase sepn. of their mol. components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, the authors combined in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liq. phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase sep. into droplets contg. distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophys. properties of the phases, particularly droplet surface tension which arises from sequence-encoded features of their macromol. components. These results suggest that phase sepn. can give rise to multilayered liqs. that may facilitate sequential RNA processing reactions in a variety of RNP bodies.
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  12. 12
    Phair, R. D.; Misteli, T. High mobility of proteins in the mammalian cell nucleus. Nature 2000, 404 (6778), 604– 9,  DOI: 10.1038/35007077
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    12
    High mobility of proteins in the mammalian cell nucleus
    Phair, Robert D.; Misteli, Tom
    Nature (London) (2000), 404 (6778), 604-609CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The mammalian cell nucleus contains numerous sub-compartments, which have been implicated in essential processes such as transcription and splicing. The mechanisms by which nuclear compartments are formed and maintained are unclear. More fundamentally, it is not known how proteins move within the cell nucleus. We have measured the kinetic properties of proteins in the nucleus of living cells using photobleaching techniques. Here we show that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus. Protein movement is independent of energy, which indicates that proteins may use a passive mechanism of movement. Proteins rapidly assoc. and dissoc. with nuclear compartments. Using kinetic modeling, we detd. residence times and steady-state fluxes of mols. in two main nuclear compartments. These data show that many nuclear proteins roam the cell nucleus in vivo and that nuclear compartments are the reflection of the steady-state assocn./dissocn. of its 'residents' with the nucleoplasmic space. Our observations have conceptual implications for understanding nuclear architecture and how nuclear processes are organized in vivo.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXis1Grs74%253D&md5=05e4181e19d87fdbbc1faa8534926024
  13. 13
    Nott, T. J.; Petsalaki, E.; Farber, P.; Jervis, D.; Fussner, E.; Plochowietz, A.; Craggs, T. D.; Bazett-Jones, D. P.; Pawson, T.; Forman-Kay, J. D.; Baldwin, A. J. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 2015, 57 (5), 936– 947,  DOI: 10.1016/j.molcel.2015.01.013
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    13
    Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles
    Nott, Timothy J.; Petsalaki, Evangelia; Farber, Patrick; Jervis, Dylan; Fussner, Eden; Plochowietz, Anne; Craggs, Timothy D.; Bazett-Jones, David P.; Pawson, Tony; Forman-Kay, Julie D.; Baldwin, Andrew J.
    Molecular Cell (2015), 57 (5), 936-947CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Cells chem. isolate mols. in compartments to both facilitate and regulate their interactions. In addn. to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that det. when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-sepd. organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temp., ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can conc. single-stranded DNA but largely exclude double-stranded DNA. We propose that phase sepn. of disordered proteins contg. weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.
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  14. 14
    Sabari, B. R.; Dall’Agnese, A.; Boija, A.; Klein, I. A.; Coffey, E. L.; Shrinivas, K.; Abraham, B. J.; Hannett, N. M.; Zamudio, A. V.; Manteiga, J. C.; Li, C. H.; Guo, Y. E.; Day, D. S.; Schuijers, J.; Vasile, E.; Malik, S.; Hnisz, D.; Lee, T. I.; Cisse, I. I.; Roeder, R. G.; Sharp, P. A.; Chakraborty, A. K.; Young, R. A. Coactivator condensation at super-enhancers links phase separation and gene control. Science 2018, 361 (6400), eaar3958,  DOI: 10.1126/science.aar3958
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    Shrinivas, K.; Sabari, B. R.; Coffey, E. L.; Klein, I. A.; Boija, A.; Zamudio, A. V.; Schuijers, J.; Hannett, N. M.; Sharp, P. A.; Young, R. A.; Chakraborty, A. K. Enhancer Features that Drive Formation of Transcriptional Condensates. Mol. Cell 2019, 75 (3), 549– 561,  DOI: 10.1016/j.molcel.2019.07.009
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    15
    Enhancer Features that Drive Formation of Transcriptional Condensates
    Shrinivas, Krishna; Sabari, Benjamin R.; Coffey, Eliot L.; Klein, Isaac A.; Boija, Ann; Zamudio, Alicia V.; Schuijers, Jurian; Hannett, Nancy M.; Sharp, Phillip A.; Young, Richard A.; Chakraborty, Arup K.
    Molecular Cell (2019), 75 (3), 549-561.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-sepd. condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site no., d., and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci detd. by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase sepn. might regulate this process.
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  16. 16
    Wippich, F.; Bodenmiller, B.; Trajkovska, M. G.; Wanka, S.; Aebersold, R.; Pelkmans, L. Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 2013, 152 (4), 791– 805,  DOI: 10.1016/j.cell.2013.01.033
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    16
    Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling
    Wippich, Frank; Bodenmiller, Bernd; Trajkovska, Maria Gustafsson; Wanka, Stefanie; Aebersold, Ruedi; Pelkmans, Lucas
    Cell (Cambridge, MA, United States) (2013), 152 (4), 791-805CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Cytosolic compartmentalization through liq.-liq. unmixing, such as the formation of RNA granules, is involved in many cellular processes and might be used to regulate signal transduction. However, specific mol. mechanisms by which liq.-liq. unmixing and signal transduction are coupled remain unknown. Here, we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of P-granule-like structures and mTORC1 signaling. DYRK3 displays a cyclic partitioning mechanism between stress granules and the cytosol via a low-complexity domain in its N-terminus and its kinase activity. When DYRK3 is inactive, it prevents stress granule dissoln. and the release of sequestered mTORC1. When DYRK3 is active, it allows stress granule dissoln., releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40. This mechanism links cytoplasmic compartmentalization via liq. phase transitions with cellular signaling.
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    Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18 (5), 285– 298,  DOI: 10.1038/nrm.2017.7
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    17
    Biomolecular condensates: organizers of cellular biochemistry
    Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A.; Rosen, Michael K.
    Nature Reviews Molecular Cell Biology (2017), 18 (5), 285-298CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. Biomol. condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to conc. proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metab., ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liq.-liq. phase sepn. driven by multivalent macromol. interactions is an important organizing principle for biomol. condensates. With this phys. framework, it is now possible to explain how the assembly, compn., phys. properties and biochem. and cellular functions of these important structures are regulated.
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    Brangwynne, C. P.; Tompa, P.; Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 2015, 11 (11), 899– 904,  DOI: 10.1038/nphys3532
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    Polymer physics of intracellular phase transitions
    Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.
    Nature Physics (2015), 11 (11), 899-904CODEN: NPAHAX; ISSN:1745-2473. (Nature Publishing Group)
    Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biol. roles, by compartmentalizing the cell to enable spatiotemporal control of biol. reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liq. droplets that form via phase sepn. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liq.-liq. phase sepn. in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equil. features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics.
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    Zumbro, E.; Alexander-Katz, A. Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation. PLoS One 2021, 16 (11), e0245405  DOI: 10.1371/journal.pone.0245405
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    19
    Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation
    Zumbro, Emiko; Alexander-Katz, Alfredo
    PLoS One (2021), 16 (11), e0245405CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Multivalent polymers are a key structural component of many biocondensates. When interacting with their cognate binding proteins, multivalent polymers such as RNA and modular proteins have been shown to influence the liq.-liq. phase sepn. (LLPS) boundary to both control condensate formation and to influence condensate dynamics after phase sepn. Much is still unknown about the function and formation of these condensed droplets, but changes in their dynamics or phase sepn. are assocd. with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's Disease. Therefore, investigation into how the structure of multivalent polymers relates to changes in biocondensate formation and maturation is essential to understanding and treating these diseases. Here, we use a coarse-grain, Brownian Dynamics simulation with reactive binding that mimics specific interactions in order to investigate the difference between non-specific and specific multivalent binding polymers. We show that non-specific binding interactions can lead to much larger changes in droplet formation at lower protein-polymer interaction energies than their specific, valence-limited counterparts. We also demonstrate the effects of solvent conditions and polymer length on phase sepn., and we present how modulating binding energy to the polymer can change the organization of a droplet in a three component system of polymer, binding protein, and solvent. Finally, we compare the effects of surface tension and polymer binding on the condensed phase dynamics, and show that both lower protein solubilities and higher attraction/affinity of the protein to the polymer result in slower droplet dynamics. This research will help to better understand exptl. systems and provides addnl. insight into how multivalent polymers can control LLPS.
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  20. 20
    Schuster, B. S.; Reed, E. H.; Parthasarathy, R.; Jahnke, C. N.; Caldwell, R. M.; Bermudez, J. G.; Ramage, H.; Good, M. C.; Hammer, D. A. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 2018, 9 (1), 2985,  DOI: 10.1038/s41467-018-05403-1
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    20
    Controllable protein phase separation and modular recruitment to form responsive membraneless organelles
    Schuster Benjamin S; Parthasarathy Ranganath; Bermudez Jessica G; Good Matthew C; Hammer Daniel A; Reed Ellen H; Jahnke Craig N; Hammer Daniel A; Caldwell Reese M; Good Matthew C; Ramage Holly
    Nature communications (2018), 9 (1), 2985 ISSN:.
    Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.
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    Garaizar, A.; Sanchez-Burgos, I.; Collepardo-Guevara, R.; Espinosa, J. R. Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid-Liquid Phase Separation. Molecules 2020, 25 (20), 4705,  DOI: 10.3390/molecules25204705
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    21
    Expansion of intrinsically disordered proteins increases the range of stability of liquid-liquid phase separation
    Garaizar, Adiran; Sanchez-Burgos, Ignacio; Collepardo-Guevara, Rosana; Espinosa, Jorge R.
    Molecules (2020), 25 (20), 4705CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Proteins contg. intrinsically disordered regions (IDRs) are ubiquitous within biomol. condensates, which are liq.-like compartments within cells formed through liq.-liq. phase sepn. (LLPS). The sequence of amino acids of a protein encodes its phase behavior, not only by establishing the patterning and chem. nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviors, including conformational switching, where they transition between alternate conformational ensembles. Using Mol. Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liq.-liq. phase behavior of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilizing multivalent protein-protein interactions by reducing steric hindrance; thereby, such conformations maximize the mol. connectivity of the condensed liq. network. Extended protein configurations promote phase sepn. regardless of whether LLPS is driven by homotypic and/or heterotypic protein-protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liq.-liq. phase behavior.
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    Flory, P. J. Principles of Polymer Chemistry; Cornell University Pres:, 1953; Vol. 1.
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    There is no corresponding record for this reference.
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    Banani, S. F.; Rice, A. M.; Peeples, W. B.; Lin, Y.; Jain, S.; Parker, R.; Rosen, M. K. Compositional Control of Phase-Separated Cellular Bodies. Cell 2016, 166 (3), 651– 663,  DOI: 10.1016/j.cell.2016.06.010
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    23
    Compositional control of phase-separated cellular bodies
    Banani, Salman F.; Rice, Allyson M.; Peeples, William B.; Lin, Yuan; Jain, Saumya; Parker, Roy; Rosen, Michael K.
    Cell (Cambridge, MA, United States) (2016), 166 (3), 651-663CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-sepd. liqs. organized by multivalent interactions among proteins and RNA mols. Although many components of various cellular bodies are known, general principles that define body compn. are lacking. Here, the authors modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold mols. formed phase-sepd. liqs. that concd. low valency client proteins. Clients partitioned differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. The compn. could switch rapidly through changes in scaffold concn. or valency. Natural PML NBs and P bodies showed analogous partitioning behavior, suggesting how their compns. could be controlled by levels of PML SUMOylation or cellular mRNA concn., resp. The data suggested a conceptual framework for considering the compn. and control thereof of cellular bodies assembled through heterotypic multivalent interactions.
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  24. 24
    Pak, C. W.; Kosno, M.; Holehouse, A. S.; Padrick, S. B.; Mittal, A.; Ali, R.; Yunus, A. A.; Liu, D. R.; Pappu, R. V.; Rosen, M. K. Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein. Mol. Cell 2016, 63 (1), 72– 85,  DOI: 10.1016/j.molcel.2016.05.042
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    24
    Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein
    Pak, Chi W.; Kosno, Martyna; Holehouse, Alex S.; Padrick, Shae B.; Mittal, Anuradha; Ali, Rustam; Yunus, Ali A.; Liu, David R.; Pappu, Rohit V.; Rosen, Michael K.
    Molecular Cell (2016), 63 (1), 72-85CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Liq.-liq. phase sepn., driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase sepn. via complex coacervation, whereby the neg. charged NICD co-assembles with pos. charged partners to form protein-rich dense liq. droplets. Mutagenesis reveals that the driving force for phase sepn. depends on the overall amino acid compn. and not the precise sequence of NICD. Instead, phase sepn. is promoted by one or more regions of high neg. charge d. and arom./hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase sepn.
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    Quiroz, F. G.; Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 2015, 14 (11), 1164– 71,  DOI: 10.1038/nmat4418
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    25
    Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers
    Quiroz, Felipe Garcia; Chilkoti, Ashutosh
    Nature Materials (2015), 14 (11), 1164-1171CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
    Proteins and synthetic polymers that undergo aq. phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behavior of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behavior, we show that the proteins can be designed to exhibit tunable lower or upper crit. soln. temp. (LCST and UCST, resp.) transitions in physiol. solns. We also show that mutation of key residues at the repeat level abolishes phase behavior or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behavior and to design novel protein polymers consisting of biol. active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behavior at the sequence level.
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    Zhang, H.; Elbaum-Garfinkle, S.; Langdon, E. M.; Taylor, N.; Occhipinti, P.; Bridges, A. A.; Brangwynne, C. P.; Gladfelter, A. S. RNA Controls PolyQ Protein Phase Transitions. Mol. Cell 2015, 60 (2), 220– 30,  DOI: 10.1016/j.molcel.2015.09.017
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    26
    RNA Controls PolyQ Protein Phase Transitions
    Zhang, Huaiying; Elbaum-Garfinkle, Shana; Langdon, Erin M.; Taylor, Nicole; Occhipinti, Patricia; Bridges, Andrew A.; Brangwynne, Clifford P.; Gladfelter, Amy S.
    Molecular Cell (2015), 60 (2), 220-230CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Compartmentalization in cells is central to the spatial and temporal control of biochem. In addn. to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liq.-liq. phase sepn. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and phys. identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiol. targets of Whi3 drive phase sepn. MRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk soln. Different mRNAs impart distinct biophys. properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophys. properties of phase-sepd. compartments.
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    Lytle, T. K.; Sing, C. E. Tuning chain interaction entropy in complex coacervation using polymer stiffness, architecture, and salt valency. Molecular Systems Design & Engineering 2018, 3 (1), 183– 196,  DOI: 10.1039/C7ME00108H
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    27
    Tuning chain interaction entropy in complex coacervation using polymer stiffness, architecture, and salt valency
    Lytle, Tyler K.; Sing, Charles E.
    Molecular Systems Design & Engineering (2018), 3 (1), 183-196CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
    Oppositely-charged polyelectrolytes can undergo a liq.-liq. phase sepn. in a salt soln., resulting in a polymer-dense 'coacervate' phase that has found use in a wide range of applications from food science to self-assembled materials. Coacervates can be tuned for specific applications by varying parameters such as salt concn. and valency, polyelectrolyte length, and polyelectrolyte identity. Recent simulation and theory has begun to clarify the role of mol. structure on coacervation phase behavior, esp. for common synthetic polyelectrolytes that exhibit high charge densities. In this manuscript, we use a combination of transfer matrix theory and Monte Carlo simulation to understand at a phys. level how a range of mol. features, in particular polymer architecture and stiffness, and salt valency can be used to design the phase diagrams of these materials. We demonstrate a phys. picture of how the underlying entropy-driven process of complex coacervation is affected by this wide range of phys. attributes.
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    Garaizar, A.; Espinosa, J. R.; Joseph, J. A.; Collepardo-Guevara, R. Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates. Sci. Rep 2022, 12 (1), 4390,  DOI: 10.1038/s41598-022-08130-2
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    28
    Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates
    Garaizar, Adiran; Espinosa, Jorge R.; Joseph, Jerelle A.; Collepardo-Guevara, Rosana
    Scientific Reports (2022), 12 (1), 4390CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
    Biomol. condensates formed by the process of liq.-liq. phase sepn. (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chem. reactions. Upon maturation, the liq.-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathol. implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a mol.-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodn. properties of biomol. condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equil. condensate has been formed, or when they accumulate slowly over time with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides-prone to forming inter-protein β-sheets-we observe that formation of inter-peptide β-sheets increases the strength of the interactions consistently with the loss of liq.-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental mol., kinetic, and thermodn. mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing.
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    Garabedian, M. V.; Wang, W.; Dabdoub, J. B.; Tong, M.; Caldwell, R. M.; Benman, W.; Schuster, B. S.; Deiters, A.; Good, M. C. Designer membraneless organelles sequester native factors for control of cell behavior. Nat. Chem. Biol. 2021, 17 (9), 998– 1007,  DOI: 10.1038/s41589-021-00840-4
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    Designer membraneless organelles sequester native factors for control of cell behavior
    Garabedian, Mikael V.; Wang, Wentao; Dabdoub, Jorge B.; Tong, Michelle; Caldwell, Reese M.; Benman, William; Schuster, Benjamin S.; Deiters, Alexander; Good, Matthew C.
    Nature Chemical Biology (2021), 17 (9), 998-1007CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
    Subcellular compartmentalization of macromols. increases flux and prevents inhibitory interactions to control biochem. reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.
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    Perdikari, T. M.; Jovic, N.; Dignon, G. L.; Kim, Y. C.; Fawzi, N. L.; Mittal, J. A predictive coarse-grained model for position-specific effects of post-translational modifications. Biophys. J. 2021, 120 (7), 1187– 1197,  DOI: 10.1016/j.bpj.2021.01.034
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    A predictive coarse-grained model for position-specific effects of post-translational
    Perdikari, Theodora Myrto; Jovic, Nina; Dignon, Gregory L.; Kim, Young C.; Fawzi, Nicolas L.; Mittal, Jeetain
    Biophysical Journal (2021), 120 (7), 1187-1197CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Biomols. undergo liq.-liq. phase sepn. (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiol. and pathol. role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the no. and position of phosphorylated and acetylated residues alter the protein's single-chain compactness and its propensity to phase sep. We show that both the no. and the position of phosphorylated threonines/serines or acetylated lysines can serve as a mol. on/off switch for phase sepn. in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, resp. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture exptl. measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase sepn. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resoln.
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    Owen, I.; Shewmaker, F. The Role of Post-Translational Modifications in the Phase Transitions of Intrinsically Disordered Proteins. Int. J. Mol. Sci. 2019, 20 (21), 5501,  DOI: 10.3390/ijms20215501
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    The role of post-translational modifications in the phase transitions of intrinsically disordered proteins
    Owen, Izzy; Shewmaker, Frank
    International Journal of Molecular Sciences (2019), 20 (21), 5501CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Advances in genomics and proteomics have revealed eukaryotic proteomes to be highly abundant in intrinsically disordered proteins that are susceptible to diverse post-translational modifications. Intrinsically disordered regions are crit. to the liq.-liq. phase sepn. that facilitates specialized cellular functions. Here, we discuss how post-translational modifications of intrinsically disordered protein segments can regulate the mol. condensation of macromols. into functional phase-sepd. complexes.
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    Söding, J.; Zwicker, D.; Sohrabi-Jahromi, S.; Boehning, M.; Kirschbaum, J. Mechanisms for Active Regulation of Biomolecular Condensates. Trends Cell Biol. 2020, 30 (1), 4– 14,  DOI: 10.1016/j.tcb.2019.10.006
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    Mechanisms for Active Regulation of Biomolecular Condensates
    Soding Johannes; Zwicker David; Kirschbaum Jan; Sohrabi-Jahromi Salma; Boehning Marc
    Trends in cell biology (2020), 30 (1), 4-14 ISSN:.
    Liquid-liquid phase separation is a key organizational principle in eukaryotic cells, on par with intracellular membranes. It allows cells to concentrate specific proteins into condensates, increasing reaction rates and achieving switch-like regulation. We propose two active mechanisms that can explain how cells regulate condensate formation and size. In both, the cell regulates the activity of an enzyme, often a kinase, that adds post-translational modifications to condensate proteins. In enrichment inhibition, the enzyme enriches in the condensate and weakens interactions, as seen in stress granules (SGs), Cajal bodies, and P granules. In localization-induction, condensates form around immobilized enzymes that strengthen interactions, as observed in DNA repair, transmembrane signaling, and microtubule assembly. These models can guide studies into the many emerging roles of biomolecular condensates.
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    Song, D.; Jo, Y.; Choi, J. M.; Jung, Y. Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions. Nat. Commun. 2020, 11 (1), 5642,  DOI: 10.1038/s41467-020-19476-4
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    Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions
    Song, Daesun; Jo, Yongsang; Choi, Jeong-Mo; Jung, Yongwon
    Nature Communications (2020), 11 (1), 5642CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liq.-liq. phase sepn., biomol. behaviors such as protein interactions inside these liq. compartments have been largely unexplored. Here we report quant. measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase sepn. conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase sepn. model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concns. inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase sepn. compartmentalization: (1) clients are selectively recruited into compartments, leading to concn. enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.
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    Zhang, H.; Zhao, R.; Tones, J.; Liu, M.; Dilley, R. L.; Chenoweth, D. M.; Greenberg, R. A.; Lampson, M. A. Nuclear body phase separation drives telomere clustering in ALT cancer cells. Mol. Biol. Cell 2020, 31 (18), 2048– 2056,  DOI: 10.1091/mbc.E19-10-0589
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    Nuclear body phase separation drives telomere clustering in ALT cancer cells
    Zhang, Huaiying; Zhao, Rongwei; Tones, Jason; Liu, Michel; Dilley, Robert L.; Chenoweth, David M.; Greenberg, Roger A.; Lampson, Michael A.
    Molecular Biology of the Cell (2020), 31 (18), 2048-2056CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    A review. Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-assocd. promyelocytic leukemia nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liq. condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chem. induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere-binding protein sumoylation nucleates APB condensation via interactions between small ubiquitin-like modifier (SUMO) and SUMO interaction motif (SIM), and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liq. properties of the condensate, as an alternative condensation chem. also induces clustering independent of sumoylation. Our findings introduce a chem. dimerization approach to manipulate phase sepn. and demonstrate how the material properties and chem. compn. of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions.
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    Wang, A.; Conicella, A. E.; Schmidt, H. B.; Martin, E. W.; Rhoads, S. N.; Reeb, A. N.; Nourse, A.; Ramirez Montero, D.; Ryan, V. H.; Rohatgi, R.; Shewmaker, F.; Naik, M. T.; Mittag, T.; Ayala, Y. M.; Fawzi, N. L. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. Embo j 2018, 37 (5), e97452  DOI: 10.15252/embj.201797452
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    Li, P.; Banjade, S.; Cheng, H. C.; Kim, S.; Chen, B.; Guo, L.; Llaguno, M.; Hollingsworth, J. V.; King, D. S.; Banani, S. F.; Russo, P. S.; Jiang, Q. X.; Nixon, B. T.; Rosen, M. K. Phase transitions in the assembly of multivalent signalling proteins. Nature 2012, 483 (7389), 336– 40,  DOI: 10.1038/nature10879
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    Phase transitions in the assembly of multivalent signalling proteins
    Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V.; King, David S.; Banani, Salman F.; Russo, Paul S.; Jiang, Qiu-Xing; Nixon, B. Tracy; Rosen, Michael K.
    Nature (London, United Kingdom) (2012), 483 (7389), 336-340CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Cells are organized on length scales ranging from angstroms to micrometers. However, the mechanisms by which angstrom-scale mol. properties are translated to micrometer-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromols. (including multidomain proteins and RNA) produce sharp liq.-liq.-demixing phase sepns., generating micrometer-sized liq. droplets in aq. soln. This macroscopic transition corresponds to a mol. transition between small complexes and large, dynamic supramol. polymers. The concns. needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biol. partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochem. regulate information throughout biol.
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    Roden, C.; Gladfelter, A. S. RNA contributions to the form and function of biomolecular condensates. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 183– 195,  DOI: 10.1038/s41580-020-0264-6
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    RNA contributions to the form and function of biomolecular condensates
    Roden, Christine; Gladfelter, Amy S.
    Nature Reviews Molecular Cell Biology (2021), 22 (3), 183-195CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
    A review. Biomol. condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liq.-liq. phase sepn. (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomol. condensation and highlight considerations for designing condensate reconstitution expts. We focus on RNA properties such as compn., length, structure, modifications and expression level. These properties can modulate the biophys. features of native condensates, including their size, shape, viscosity, liquidity, surface tension and compn. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be detd. by RNA. Finally, we discuss the multifaceted cellular functions of biomol. condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific mols., and buffering noise and responding to stress.
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    Tejedor, A. R.; Garaizar, A.; Ramírez, J.; Espinosa, J. R. RNA modulation of transport properties and stability in phase-separated condensates. Biophys. J. 2021, 120 (23), 5169– 5186,  DOI: 10.1016/j.bpj.2021.11.003
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    'RNA modulation of transport properties and stability in phase-separated condensates
    Tejedor, Andres R.; Garaizar, Adiran; Ramirez, Jorge; Espinosa, Jorge R.
    Biophysical Journal (2021), 120 (23), 5169-5186CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissoln. of phase-sepd. condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chem.-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomol. condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concn. By characterizing the phase diagram, key mol. interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase sepn. at low concn. as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concn., it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concn. as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concn., but barely modify protein self-diffusion which mainly depends on RNA concn. and on the effect RNA has on droplet d. On the whole, our work rationalizes the different routes by which RNA can regulate phase sepn. and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.
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    Li, N. K.; Roberts, S.; Quiroz, F. G.; Chilkoti, A.; Yingling, Y. G. Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides. Biomacromolecules 2018, 19 (7), 2496– 2505,  DOI: 10.1021/acs.biomac.8b00099
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    Sequence directionality dramatically affects LCST behavior of elastin-like polypeptides
    Li, Nan K.; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G.
    Biomacromolecules (2018), 19 (7), 2496-2505CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Elastin-like polypeptides (ELP) exhibit an inverse temp. transition or lower crit. soln. temp. (LCST) transition phase behavior in aq. solns. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, CD (CD) measurements, and all-atom mol. dynamics (MD) simulations to gain a mol. understanding of mechanism that controls hysteretic phase behavior. It was shown exptl. that both poly(VPGVG) and poly(VGPVG) undergo a transition from sol. to insol. in aq. soln. upon heating above the transition temp. (Tt). However, poly(VPGVG) resolubilizes upon cooling below its Tt, whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the Tt. The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, esp. valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above Tt, where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the obsd. thermal hysteresis for poly(VGPVG).
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    Dzuricky, M.; Rogers, B. A.; Shahid, A.; Cremer, P. S.; Chilkoti, A. De novo engineering of intracellular condensates using artificial disordered proteins. Nat. Chem. 2020, 12 (9), 814– 825,  DOI: 10.1038/s41557-020-0511-7
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    De novo engineering of intracellular condensates using artificial disordered proteins
    Dzuricky, Michael; Rogers, Bradley A.; Shahid, Abdulla; Cremer, Paul S.; Chilkoti, Ashutosh
    Nature Chemistry (2020), 12 (9), 814-825CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    Phase sepn. of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, the authors systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different mol. wts. and arom. content, which exhibit variable condensate satn. concns. and temp. cloud points in vitro and in cells. The authors created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the mol. wt. of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-sepn.-mediated control of biol. function in living cells.
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    Basheer, A.; Shahid, S.; Kang, M. J.; Lee, J. H.; Lee, J. S.; Lim, D. W. Switchable Self-Assembly of Elastin- and Resilin-Based Block Copolypeptides with Converse Phase Transition Behaviors. ACS Appl. Mater. Interfaces 2021, 13 (21), 24385– 24400,  DOI: 10.1021/acsami.1c00676
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    Switchable Self-Assembly of Elastin- and Resilin-Based Block Copolypeptides with Converse Phase Transition Behaviors
    Basheer, Aamna; Shahid, Shahzaib; Kang, Min Jung; Lee, Jae Hee; Lee, Jae Sang; Lim, Dong Woo
    ACS Applied Materials & Interfaces (2021), 13 (21), 24385-24400CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Self-assembly of thermally responsive polypeptides into unique nanostructures offers intriguing attributes including dynamic phys. dimensions, biocompatibility, and biodegradability for the smart bio-nanomaterials. As elastin-based polypeptide (EBP) fusion proteins with lower crit. soln. temp. (LCST) are studied as drug delivery systems, EBP block copolypeptides with the resilin-based polypeptide (RBP) displaying an upper crit. soln. temp. (UCST) have been of great interest. In this study, we report thermally triggered, dynamic self-assembly of EBP- and RBP-based diblock copolypeptides into switched nanostructures with reversibility under physiol. conditions. Mol. DNA clones encoding for the EBP-RBP diblocks at different block length ratios were biosynthesized via recursive directional ligation and overexpressed, followed by nonchromatog. purifn. by inverse transition cycling. Genetically engineered diblock copolypeptides composed of the EBP with an LCST and the RBP with a UCST showed converse phase transition behaviors with both a distinct LCST and a distinct UCST (LCST < UCST). As temp. increased, three phases of these EBP-RBP diblocks were obsd.: (1) self-assembled micelles or vesicles below both LCST and UCST, (2) whole aggregates above LCST and below UCST, and (3) reversed micelles above both LCST and UCST. In conclusion, these stimuli-triggered, dynamic protein-based nanostructures are promising for advanced drug delivery systems, regenerative medicine, and biomedical nanotechnol.
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    Reed, E. H.; Hammer, D. A. Redox sensitive protein droplets from recombinant oleosin. Soft Matter 2018, 14 (31), 6506– 6513,  DOI: 10.1039/C8SM01047A
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    Redox sensitive protein droplets from recombinant oleosin
    Reed, Ellen H.; Hammer, Daniel A.
    Soft Matter (2018), 14 (31), 6506-6513CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
    Protein engineering enables the creation of materials with designer functionality and tailored responsiveness. Here, we design a protein with two control motifs for its phase sepn. into micron sized liq. droplets - one driven by a hydrophobic domain and the other by oxidn. of a disulfide bond. Our work is based on the plant surfactant protein, oleosin, which has a hydrophobic domain but no cysteines. Oleosin phase separates to form liq. droplets below a crit. temp. akin to many naturally occurring membrane-less organelles. Sequence mutations are made to introduce a cysteine residue into oleosin. The addn. of a cysteine causes phase sepn. at a lower concn. and increases the phase transition temp. Adding a reducing agent to phase-sepd., cysteine-contg. oleosin rapidly dissolves the droplets. The transition temp. is tuned by varying the location of the cysteine or by blending the parent cysteine-less mol. with the cysteine-contg. mutant. This provides a novel way to control protein droplet formation and dissoln. We envision this work having applications as a system for the release of a protein or drug with engineered sensitivity to reducing conditions and as a mimic of membrane-less organelles in synthetic protocells.
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  43. 43
    Costa, S. A.; Simon, J. R.; Amiram, M.; Tang, L.; Zauscher, S.; Brustad, E. M.; Isaacs, F. J.; Chilkoti, A. Photo-Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano- to Microgels of Intrinsically Disordered Polypeptides. Adv. Mater. 2018, 30 (5), 1704878,  DOI: 10.1002/adma.201704878
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    Agouridas, V.; El Mahdi, O.; Diemer, V.; Cargoët, M.; Monbaliu, J. M.; Melnyk, O. Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations. Chem. Rev. 2019, 119 (12), 7328– 7443,  DOI: 10.1021/acs.chemrev.8b00712
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    Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations
    Agouridas, Vangelis; El Mahdi, Ouafaa; Diemer, Vincent; Cargoet, Marine; Monbaliu, Jean-Christophe M.; Melnyk, Oleg
    Chemical Reviews (Washington, DC, United States) (2019), 119 (12), 7328-7443CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The native chem. ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnol. research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and phys. chem. studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O,S-, N,S-, or N,Se-acyl shift systems as thioester or selenoester surrogates and discusses the special mol. features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N,S-acyl migrations and selective dechalcogenation reactions.
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    Keeble, A. H.; Howarth, M. Power to the protein: enhancing and combining activities using the Spy toolbox. Chem. Sci. 2020, 11 (28), 7281– 7291,  DOI: 10.1039/D0SC01878C
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    Power to the protein: enhancing and combining activities using the Spy toolbox
    Keeble, Anthony H.; Howarth, Mark
    Chemical Science (2020), 11 (28), 7281-7291CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    A review. Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein anal. or application. Here we review SpyTag technol., now used in hundreds of publications or patents, and its potential for detecting and controlling protein behavior. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purifn. of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. SpyTag/SpyCatcher is mech. stable, so is widely used for investigating protein folding and force sensitivity. A toolbox of scaffolds allows SpyTag-fusions to be assembled into defined multimers, from dimers to 180-mers, or unlimited 1D, 2D or 3D networks. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. For enzymes, Spy technol. has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors.
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    Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. Characterization of the FKBP.rapamycin.FRB ternary complex. J. Am. Chem. Soc. 2005, 127 (13), 4715– 21,  DOI: 10.1021/ja043277y
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    46
    Characterization of the FKBP·Rapamycin·FRB Ternary Complex
    Banaszynski, Laura A.; Liu, Corey W.; Wandless, Thomas J.
    Journal of the American Chemical Society (2005), 127 (13), 4715-4721CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough anal. of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP·rapamycin·FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Anal. of the data shows that rapamycin binds to FRB with moderate affinity (Kd = 26±0.8 μM). The FKBP12·rapamycin complex, however, binds to FRB 2000-fold more tightly (Kd = 12±0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiol. conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.
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  47. 47
    Caldwell, R. M.; Bermudez, J. G.; Thai, D.; Aonbangkhen, C.; Schuster, B. S.; Courtney, T.; Deiters, A.; Hammer, D. A.; Chenoweth, D. M.; Good, M. C. Optochemical Control of Protein Localization and Activity within Cell-like Compartments. Biochemistry 2018, 57 (18), 2590– 2596,  DOI: 10.1021/acs.biochem.8b00131
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    Optochemical control of protein localization and activity within cell-like compartments
    Caldwell, Reese M.; Bermudez, Jessica G.; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S.; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A.; Chenoweth, David M.; Good, Matthew C.
    Biochemistry (2018), 57 (18), 2590-2596CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    We report inducible dimerization strategies for controlling protein positioning, enzymic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compd., we demonstrate small mol. and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biol. processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a crit. step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
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    Ballister, E. R.; Aonbangkhen, C.; Mayo, A. M.; Lampson, M. A.; Chenoweth, D. M. Localized light-induced protein dimerization in living cells using a photocaged dimerizer. Nat. Commun. 2014, 5, 5475,  DOI: 10.1038/ncomms6475
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    Localized light-induced protein dimerization in living cells using a photocaged dimerizer
    Ballister Edward R; Mayo Alyssa M; Lampson Michael A; Aonbangkhen Chanat; Chenoweth David M
    Nature communications (2014), 5 (), 5475 ISSN:.
    Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein-protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.
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    Inobe, T.; Nukina, N. Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins. J. Biosci Bioeng 2016, 122 (1), 40– 6,  DOI: 10.1016/j.jbiosc.2015.12.004
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    Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins
    Inobe, Tomonao; Nukina, Nobuyuki
    Journal of Bioscience and Bioengineering (2016), 122 (1), 40-46CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)
    Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addn. of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biol. applications.
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    Reed, E. H.; Schuster, B. S.; Good, M. C.; Hammer, D. A. SPLIT: Stable Protein Coacervation Using a Light Induced Transition. ACS Synth. Biol. 2020, 9 (3), 500– 507,  DOI: 10.1021/acssynbio.9b00503
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    50
    SPLIT: Stable Protein Coacervation Using a Light Induced Transition
    Reed, Ellen H.; Schuster, Benjamin S.; Good, Matthew C.; Hammer, Daniel A.
    ACS Synthetic Biology (2020), 9 (3), 500-507CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Protein coacervates serve as hubs to conc. and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiol. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein contg. a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase sepn. using light.
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  51. 51
    Schuster, B. S.; Dignon, G. L.; Tang, W. S.; Kelley, F. M.; Ranganath, A. K.; Jahnke, C. N.; Simpkins, A. G.; Regy, R. M.; Hammer, D. A.; Good, M. C.; Mittal, J. Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (21), 11421– 11431,  DOI: 10.1073/pnas.2000223117
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    Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior
    Schuster, Benjamin S.; Dignon, Gregory L.; Tang, Wai Shing; Kelley, Fleurie M.; Ranganath, Aishwarya Kanchi; Jahnke, Craig N.; Simpkins, Alison G.; Regy, Roshan Mammen; Hammer, Daniel A.; Good, Matthew C.; Mittal, Jeetain
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (21), 11421-11431CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Phase sepn. of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize mols. intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase sepn. is crit. for understanding physiol. roles and pathol. consequences of biomol. condensation, as well as for harnessing phase sepn. for applications in bioinspired materials design. To expand the authors' knowledge of sequence determinants of IDP phase sepn., the authors characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase sepn. and a key component of P granules. Based on a predictive coarse-grained IDP model, the authors identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase sepn. in vitro and in vivo. The authors detd. the effects of charge patterning on phase behavior through sequence shuffling. The authors designed sequences with significantly increased phase sepn. propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under neg. selection to moderate this mode of interaction. The authors measured the contributions of tyrosine and arginine residues to phase sepn. exptl. through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, despite these sequence perturbations, the RGG-derived condensates remain liq.-like. Together, these studies advance the authors' fundamental understanding of key biophys. principles and sequence features important to phase sepn.
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    Apostolovic, B.; Danial, M.; Klok, H. A. Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem. Soc. Rev. 2010, 39 (9), 3541– 75,  DOI: 10.1039/b914339b
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    Coiled coils: Attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials
    Apostolovic, Bojana; Danial, Maarten; Klok, Harm-Anton
    Chemical Society Reviews (2010), 39 (9), 3541-3575CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The coiled-coil is a superhelical protein structural motif that consists of 2 or more α-helical peptides that are wrapped around each other in superhelical fashion. Coiled-coils are among the most ubiquitous folding motifs found in proteins and have not only been identified in structural proteins but also play an important role in various intracellular regulation processes as well as membrane fusion. The aim of this crit. review is to highlight the potential of coiled coil peptide sequences for the development of self-assembled, responsive and/or bioactive materials. After a short historical overview outlining the discovery of this protein folding motif, the article briefly discusses naturally occurring coiled-coils. After that, the basic rules, which have been established to date for the design of coiled-coils is briefly summarized followed by a presentation of several classes of coiled-coils, which may represent interesting candidates for the development of novel self-assembled, responsive and/or bioactive materials. This crit. review ends with a section that summarizes the different coiled-coil-based (hybrid) materials that have been reported to date and which hopefully will help to stimulate further work to explore the full potential of this unique class of protein folding motifs for the development of novel self-assembled, responsive and/or bioactive materials.
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  53. 53
    Fletcher, J. M.; Boyle, A. L.; Bruning, M.; Bartlett, G. J.; Vincent, T. L.; Zaccai, N. R.; Armstrong, C. T.; Bromley, E. H.; Booth, P. J.; Brady, R. L.; Thomson, A. R.; Woolfson, D. N. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology. ACS Synth. Biol. 2012, 1 (6), 240– 50,  DOI: 10.1021/sb300028q
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    53
    A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology
    Fletcher, Jordan M.; Boyle, Aimee L.; Bruning, Marc; Bartlett, Gail J.; Vincent, Thomas L.; Zaccai, Nathan R.; Armstrong, Craig T.; Bromley, Elizabeth H. C.; Booth, Paula J.; Brady, R. Leo; Thomson, Andrew R.; Woolfson, Derek N.
    ACS Synthetic Biology (2012), 1 (6), 240-250CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Protein engineering, chem. biol., and synthetic biol. would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and anal. of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in soln. by circular-dichroism spectroscopy and anal. ultracentrifugation, and their structures were detd. by X-ray crystallog. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed exptl. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chem. and synthetic biol. To help with such endeavors, we introduce Pcomp, an online registry of peptide components for protein-design and synthetic-biol. applications.
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    Majerle, A.; Hadži, S.; Aupič, J.; Satler, T.; Lapenta, F.; Strmšek, Ž.; Lah, J.; Loris, R.; Jerala, R. A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (17), e2021899118  DOI: 10.1073/pnas.2021899118
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    A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures
    Majerle, Andreja; Hadzi, San; Aupic, Jana; Satler, Tadej; Lapenta, Fabio; Strmsek, Ziga; Lah, Jurij; Loris, Remy; Jerala, Roman
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (17), e2021899118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that det. the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong pos. cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.
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    Georgoulia, P. S.; Bjelic, S. Prediction of Protein-Protein Binding Interactions in Dimeric Coiled Coils by Information Contained in Folding Energy Landscapes. Int. J. Mol. Sci. 2021, 22 (3), 1368,  DOI: 10.3390/ijms22031368
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    Prediction of protein-protein binding interactions in dimeric coiled coils by information contained in folding energy landscapes
    Georgoulia, Panagiota S.; Bjelic, Sinisa
    International Journal of Molecular Sciences (2021), 22 (3), 1368CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    Coiled coils represent the simplest form of a complex formed between two interacting protein partners. Their extensive study has led to the development of various methods aimed towards the investigation and design of complex forming interactions. Despite the progress that has been made to predict the binding affinities for protein complexes, and specifically those tailored towards coiled coils, many challenges still remain. In this work, we explore whether the information contained in dimeric coiled coil folding energy landscapes can be used to predict binding interactions. Using the published SYNZIP dataset, we start from the amino acid sequence, to simultaneously fold and dock approx. 1000 coiled coil dimers. Assessment of the folding energy landscapes showed that a model based on the calcd. no. of clusters for the lowest energy structures displayed a signal that correlates with the exptl. detd. protein interactions. Although the revealed correlation is weak, we show that such correlation exists; however, more work remains to establish whether further improvements can be made to the presented model.
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    Thompson, K. E.; Bashor, C. J.; Lim, W. A.; Keating, A. E. SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth. Biol. 2012, 1 (4), 118– 29,  DOI: 10.1021/sb200015u
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    56
    SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo Specifications of Heterospecific Coiled-Coil Interaction Domains
    Thompson, Kenneth Evan; Bashor, Caleb J.; Lim, Wendell A.; Keating, Amy E.
    ACS Synthetic Biology (2012), 1 (4), 118-129CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The synthetic biol. toolkit contains a growing no. of parts for regulating transcription and translation, but very few that can be used to control protein assocn. Here the authors report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. The authors present biophys. anal. of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the no. of available protein interaction parts for synthetic biol. and should facilitate a wide range of mol. engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].
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    Lebar, T.; Lainšček, D.; Merljak, E.; Aupič, J.; Jerala, R. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat. Chem. Biol. 2020, 16 (5), 513– 519,  DOI: 10.1038/s41589-019-0443-y
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    57
    A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells
    Lebar, Tina; Lainscek, Dusko; Merljak, Estera; Aupic, Jana; Jerala, Roman
    Nature Chemical Biology (2020), 16 (5), 513-519CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biol. systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small mol.-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biol.
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  58. 58
    Shin, Y.; Berry, J.; Pannucci, N.; Haataja, M. P.; Toettcher, J. E.; Brangwynne, C. P. Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Cell 2017, 168 (1–2), 159– 171,  DOI: 10.1016/j.cell.2016.11.054
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    58
    Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets
    Shin, Yongdae; Berry, Joel; Pannucci, Nicole; Haataja, Mikko P.; Toettcher, Jared E.; Brangwynne, Clifford P.
    Cell (Cambridge, MA, United States) (2017), 168 (1-2), 159-171.e14CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liq.-like ribonucleoprotein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiol. and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concn. threshold, these constructs undergo light-activated phase sepn., forming spatiotemporally definable liq. optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiol. phase transitions but also their link to pathol. aggregates.
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  59. 59
    Bracha, D.; Walls, M. T.; Wei, M. T.; Zhu, L.; Kurian, M.; Avalos, J. L.; Toettcher, J. E.; Brangwynne, C. P. Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds. Cell 2018, 175 (6), 1467– 1480,  DOI: 10.1016/j.cell.2018.10.048
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    59
    Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds
    Bracha, Dan; Walls, Mackenzie T.; Wei, Ming-Tzo; Zhu, Lian; Kurian, Martin; Avalos, Jose L.; Toettcher, Jared E.; Brangwynne, Clifford P.
    Cell (Cambridge, MA, United States) (2018), 175 (6), 1467-1480.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Liq.-liq. phase sepn. plays a key role in the assembly of diverse intracellular structures. However, the biophys. principles by which phase sepn. can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quant. light-controlled tunability to map full intracellular phase diagrams, which dictate the concns. at which phase sepn. occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both expts. and simulations show that while intracellular concns. may be insufficient for global phase sepn., sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase sepn. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates assocd. with diverse biol. processes.
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  60. 60
    Wei, S. P.; Qian, Z. G.; Hu, C. F.; Pan, F.; Chen, M. T.; Lee, S. Y.; Xia, X. X. Formation and functionalization of membraneless compartments in Escherichia coli. Nat. Chem. Biol. 2020, 16 (10), 1143– 1148,  DOI: 10.1038/s41589-020-0579-9
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    60
    Formation and functionalization of membraneless compartments in Escherichia coli
    Wei, Shao-Peng; Qian, Zhi-Gang; Hu, Chun-Fei; Pan, Fang; Chen, Meng-Ting; Lee, Sang Yup; Xia, Xiao-Xia
    Nature Chemical Biology (2020), 16 (10), 1143-1148CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Membraneless organelles formed by liq.-liq. phase sepn. of proteins or nucleic acids are involved in diverse biol. processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liq. protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiol. crowding environments of E. coli revealed that the condensates are formed via liq.-liq. phase sepn. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochem. reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biol.
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    Zhao, E. M.; Suek, N.; Wilson, M. Z.; Dine, E.; Pannucci, N. L.; Gitai, Z.; Avalos, J. L.; Toettcher, J. E. Light-based control of metabolic flux through assembly of synthetic organelles. Nat. Chem. Biol. 2019, 15 (6), 589– 597,  DOI: 10.1038/s41589-019-0284-8
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    61
    Light-based control of metabolic flux through assembly of synthetic organelles
    Zhao, Evan M.; Suek, Nathan; Wilson, Maxwell Z.; Dine, Elliot; Pannucci, Nicole L.; Gitai, Zemer; Avalos, Jose L.; Toettcher, Jared E.
    Nature Chemical Biology (2019), 15 (6), 589-597CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    To maximize a desired product, metabolic engineers typically express enzymes to high, const. levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabs. in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concn. of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
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  62. 62
    Aonbangkhen, C.; Zhang, H.; Wu, D. Z.; Lampson, M. A.; Chenoweth, D. M. Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer. J. Am. Chem. Soc. 2018, 140 (38), 11926– 11930,  DOI: 10.1021/jacs.8b07753
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    62
    Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer
    Aonbangkhen, Chanat; Zhang, Huaiying; Wu, Daniel Z.; Lampson, Michael A.; Chenoweth, David M.
    Journal of the American Chemical Society (2018), 140 (38), 11926-11930CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Many dynamic biol. processes are regulated by protein-protein interactions and protein localization. Exptl. techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chem. induced dimerization (CID) of proteins. CID has been used to study several cellular events, esp. cell signaling networks, which are often reversible. However, chem. dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resoln. are currently limited. Herein, we present a novel chem. inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this mol. by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biol. questions on subcellular levels by controlling protein-protein interactions.
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    Haruki, H.; Nishikawa, J.; Laemmli, U. K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 2008, 31 (6), 925– 32,  DOI: 10.1016/j.molcel.2008.07.020
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    The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes
    Haruki, Hirohito; Nishikawa, Junichi; Laemmli, Ulrich K.
    Molecular Cell (2008), 31 (6), 925-932CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
    The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addn. of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addn., when examd. functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biol. to dissect the function of individual or gene pairs in synthetic, lethal situations.
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    Woods, B.; Kuo, C. C.; Wu, C. F.; Zyla, T. R.; Lew, D. J. Polarity establishment requires localized activation of Cdc42. J. Cell Biol. 2015, 211 (1), 19– 26,  DOI: 10.1083/jcb.201506108
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    Polarity establishment requires localized activation of Cdc42
    Woods, Benjamin; Kuo, Chun-Chen; Wu, Chi-Fang; Zyla, Trevin R.; Lew, Daniel J.
    Journal of Cell Biology (2015), 211 (1), 19-26CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the front of the cell. The high local concn. of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve pos. feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct pos. feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system.
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    Langan, R. A.; Boyken, S. E.; Ng, A. H.; Samson, J. A.; Dods, G.; Westbrook, A. M.; Nguyen, T. H.; Lajoie, M. J.; Chen, Z.; Berger, S.; Mulligan, V. K.; Dueber, J. E.; Novak, W. R. P.; El-Samad, H.; Baker, D. De novo design of bioactive protein switches. Nature 2019, 572 (7768), 205– 210,  DOI: 10.1038/s41586-019-1432-8
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    De novo design of bioactive protein switches
    Langan, Robert A.; Boyken, Scott E.; Ng, Andrew H.; Samson, Jennifer A.; Dods, Galen; Westbrook, Alexandra M.; Nguyen, Taylor H.; Lajoie, Marc J.; Chen, Zibo; Berger, Stephanie; Mulligan, Vikram Khipple; Dueber, John E.; Novak, Walter R. P.; El-Samad, Hana; Baker, David
    Nature (London, United Kingdom) (2019), 572 (7768), 205-210CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Allosteric regulation of protein function is widespread in biol., but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramol. interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degrdn. or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biol. and cell engineering.
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  68. 68
    Ng, A. H.; Nguyen, T. H.; Gómez-Schiavon, M.; Dods, G.; Langan, R. A.; Boyken, S. E.; Samson, J. A.; Waldburger, L. M.; Dueber, J. E.; Baker, D.; El-Samad, H. Modular and tunable biological feedback control using a de novo protein switch. Nature 2019, 572 (7768), 265– 269,  DOI: 10.1038/s41586-019-1425-7
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    68
    Modular and tunable biological feedback control using a de novo protein switch
    Ng, Andrew H.; Nguyen, Taylor H.; Gomez-Schiavon, Mariana; Dods, Galen; Langan, Robert A.; Boyken, Scott E.; Samson, Jennifer A.; Waldburger, Lucas M.; Dueber, John E.; Baker, David; El-Samad, Hana
    Nature (London, United Kingdom) (2019), 572 (7768), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technol.8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here the authors leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signaling pathways and synthetic gene circuits. The authors first generate synthetic neg. and pos. feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signaling mols., illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. The authors next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behavior in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biol.10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnol. and therapeutic applications.
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    Ljubetič, A.; Lapenta, F.; Gradišar, H.; Drobnak, I.; Aupič, J.; Strmšek, Ž.; Lainšček, D.; Hafner-Bratkovič, I.; Majerle, A.; Krivec, N.; Benčina, M.; Pisanski, T.; Veličković, T.; Round, A.; Carazo, J. M.; Melero, R.; Jerala, R. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 2017, 35 (11), 1094– 1101,  DOI: 10.1038/nbt.3994
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    Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo
    Ljubetic, Ajasja; Lapenta, Fabio; Gradisar, Helena; Drobnak, Igor; Aupic, Jana; Strmsek, Ziga; Lainscek, Dusko; Hafner-Bratkovic, Iva; Majerle, Andreja; Krivec, Nusa; Bencina, Mojca; Pisanski, Tomaz; Velickovic, Tanja Cirkovic; Round, Adam; Carazo, Jose Maria; Melero, Roberto; Jerala, Roman
    Nature Biotechnology (2017), 35 (11), 1094-1101CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest contg. >700 amino-acid residues and measuring 11 nm in diam. Their stability and folding kinetics were similar to those of natural proteins. Soln. small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophys. anal. confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.
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    Seuring, J.; Agarwal, S. Polymers with upper critical solution temperature in aqueous solution. Macromol. Rapid Commun. 2012, 33 (22), 1898– 920,  DOI: 10.1002/marc.201200433
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    Polymers with Upper Critical Solution Temperature in Aqueous Solution
    Seuring, Jan; Agarwal, Seema
    Macromolecular Rapid Communications (2012), 33 (22), 1898-1920CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. This review focuses on polymers with upper crit. soln. temp. (UCST) in water or electrolyte soln. and provides a detailed survey of the yet few existing examples. A guide for synthetic chemists for the design of novel UCST polymers is presented and possible handles to tune the phase transition temp., sharpness of transition, hysteresis, and effectiveness of phase sepn. are discussed. This review tries to answer the question why polymers with UCST remained largely underrepresented in academic as well as applied research and what requirements have to be fulfilled to make these polymers suitable for the development of smart materials with a pos. thermoresponse.
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    Dignon, G. L.; Zheng, W.; Mittal, J. Simulation methods for liquid-liquid phase separation of disordered proteins. Curr. Opin Chem. Eng. 2019, 23, 92– 98,  DOI: 10.1016/j.coche.2019.03.004
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    Simulation methods for liquid-liquid phase separation of disordered proteins
    Dignon Gregory L; Mittal Jeetain; Zheng Wenwei
    Current opinion in chemical engineering (2019), 23 (), 92-98 ISSN:2211-3398.
    Liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and other biomolecules is a highly complex but robust process used by living systems. Drawing inspiration from biology, phase separating proteins have been successfully utilized for promising applications in fields of materials design and drug delivery. These protein-based materials are advantageous due to the ability to finely tune their stimulus-responsive phase behavior and material properties, and the ability to encode biologically active motifs directly into the sequence. The number of possible protein sequences is virtually endless, which makes sequence-based design a rather daunting task, but also attractive due to the amount of control coming from exploration of this variable space. The use of computational methods in this field of research have come to the aid in several aspects, including interpreting experimental results, identifying important structural features and molecular mechanisms capable of explaining the phase behavior, and ultimately providing predictive frameworks for rational design of protein sequences. Here we provide an overview of computational studies focused on phase separating biomolecules and the tools that are available to researchers interested in this topic.
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    Chou, H. Y.; Aksimentiev, A. Single-Protein Collapse Determines Phase Equilibria of a Biological Condensate. J. Phys. Chem. Lett. 2020, 11 (12), 4923– 4929,  DOI: 10.1021/acs.jpclett.0c01222
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    72
    Single-Protein Collapse Determines Phase Equilibria of a Biological Condensate
    Chou, Han-Yi; Aksimentiev, Aleksei
    Journal of Physical Chemistry Letters (2020), 11 (12), 4923-4929CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Recent advances in microscopy of living cells have established membraneless organelles as crit. elements of diverse biol. processes. The body of exptl. work suggests that formation of such organelles is driven by liq.-liq. phase sepn., a phys. process that has been studied extensively for both simple liqs. and mixts. of polymers. Here, we combine mol. dynamics simulations with polymer theory to show that the thermodn. behavior of one particular biomol. condensate-fused in sarcoma (FUS)-can be quant. accounted for at the level of the chain collapse theory. First, we show that a particle-based mol. dynamics model can reproduce known phase sepn. properties of a FUS condensate, including its crit. concn. and susceptibility to mutations. Next, we obtain a polymer physics representation of a FUS condensate by examg. the behavior of a single FUS protein as a function of temp. We use the chain collapse theory to det. the thermodn. properties of the condensate and to characterize changes in the single-chain conformation at the onset of phase sepn. Altogether, our findings suggest that the phase behavior of FUS condensates can be explained by the properties of individual FUS proteins and that the change in the FUS conformation is the main force driving for the phase sepn.
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    Karginov, A. V.; Zou, Y.; Shirvanyants, D.; Kota, P.; Dokholyan, N. V.; Young, D. D.; Hahn, K. M.; Deiters, A. Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J. Am. Chem. Soc. 2011, 133 (3), 420– 3,  DOI: 10.1021/ja109630v
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    73
    Light Regulation of Protein Dimerization and Kinase Activity in Living Cells using Photocaged Rapamycin and Engineered FKBP
    Karginov, Andrei V.; Zou, Yan; Shirvanyants, David; Kota, Pradeep; Dokholyan, Nikolay V.; Young, Douglas D.; Hahn, Klaus M.; Deiters, Alexander
    Journal of the American Chemical Society (2011), 133 (3), 420-423CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We developed a new system for light-induced protein dimerization in living cells using a photocaged analog of rapamycin together with an engineered rapamycin binding domain. Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
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  74. 74
    Heidenreich, M.; Georgeson, J. M.; Locatelli, E.; Rovigatti, L.; Nandi, S. K.; Steinberg, A.; Nadav, Y.; Shimoni, E.; Safran, S. A.; Doye, J. P. K.; Levy, E. D. Designer protein assemblies with tunable phase diagrams in living cells. Nat. Chem. Biol. 2020, 16 (9), 939– 945,  DOI: 10.1038/s41589-020-0576-z
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    74
    Designer protein assemblies with tunable phase diagrams in living cells
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    Nature Chemical Biology (2020), 16 (9), 939-945CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
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Cited By

This article is cited by 3 publications.

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  2. Karthik Nadendla, Grant G. Simpson, Julie Becher, Toby Journeaux, Mar Cabeza-Cabrerizo, Gonçalo J. L. Bernardes. Strategies for Conditional Regulation of Proteins. JACS Au 2023, 3 (2) , 344-357. https://doi.org/10.1021/jacsau.2c00654
  3. Thijs W. van Veldhuisen, Wiggert J. Altenburg, Madelief A. M. Verwiel, Lenne J. M. Lemmens, Alexander F. Mason, Maarten Merkx, Luc Brunsveld, Jan C. M. van Hest. Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells. Advanced Materials 2023, 35 (29) https://doi.org/10.1002/adma.202300947
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  • Abstract

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    Figure 1

    Figure 1. Tuning Csat for condensation using noncovalent RGG multimerization via coiled-coil pairs. (A) Schematic of assembly of higher order RGG disordered polypeptides by genetic fusion to cognate pairs of helical coiled coils. In OFF (monomer) state, monomer concentrations should be below their Csat, and in ON (dimer) state, higher polymer valency (length) lowers Csat such that the dimer condenses into liquid-like droplets. (B, D, and F) Representative fluorescence microscopy images of condensates formed using 4 μM RGG monomer concentrations, 150 mM salt, pH 8.5. (B) images of SZ1-RGG, RGG-SZ2, and a mixture of SZ1-RGG and RGG-SZ2. (C) Turbidity measurements at A600 for 6 μM concentration of the control RGG-RGG dimer, indicated monomers, or mixtures of monomers, over a range of temperatures. (D) Images of P3-RGG, RGG-P4, and combination of P3-RGG and RGG-P4. (E) Turbidity measurement using a 6 μM concentration of the indicated control, monomer, and mixed monomer over a range of temperatures. (F) Representative images of RGG-P5, RGG-P6, and a mixture of RGG-P5 and RGG-P5. (G) Turbidity measurement using a 6 μM concentration of the indicated control dimer, monomer, and mixed monomer over a range of temperatures.

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    Figure 2

    Figure 2. Temporal control of IDR multimerization and phase separation in vitro. (A) Schematic representation for chemogenic dimerization of RGG polypeptides to form mesoscale liquid-like protein condensates. (B) Representative images of liquid droplet formation through increased domain valency upon addition of dimerizer, Rap. Recombinant RGG-FKBP and RGG-FRB proteins, in the absence of Rap, do not form condensates in a buffer containing 10 μM protein and 0.2 μM tracer (RGG-GFP-RGG). Scale bar, 10 μm. Addition of Rap to the reaction rapidly induces dimerization, causing condensate formation similar to the RGG-RGG constitutive dimer. (C and D) Quantitation of the kinetics of droplet formation upon equimolar addition of Rap. Average of three independent trials. Shaded area, StDev. (E) Kinetics of solution clouding after addition of dimerizer in spectrophotometric turbidity assays; average of three experiments area shown. (F) Phase transition temperature measured by turbidity assay shows induced dimerization of RGG-FKBP and RGG-FRB with Rap shifts the cloud point to higher temperatures, similar to constitutive RGG-RGG dimer; average of three experiments. (G) Representative images from photobleaching and recovery of condensates composed of Rap-mediated RGG-FKBP/RGG-FRB dimers marked by 0.2 μM RGG-GFP-RGG tracer. Scale bar 5 μm. (H) Quantification of FRAP indicating similar recovery kinetics of Rap-induced vs constitutive RGG-RGG dimers. n = 30 condensates from two independent trials.

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    Figure 3

    Figure 3. Optochemical regulation of IDR condensation. (A) Chemical structure of photocaged rapamycin, dRap. (B) Schematic of approach: RGG domains fused to FKBP or FRB tags do not dimerize in the presence of dRap. Upon illumination, dRap is uncaged to Rap, resulting in dimerization of RGG-FKBP and RGG-FRB. (C) Pre- and postillumination images of water-in-oil emulsions, stabilized by Cithrol DPHS surfactant, encapsulating 10 μM each of RGG-FKBP and RGG-FRB, 0.2 μM RGG-GFP-RGG as a fluorescent tracer, and 5 μM dRap. Dotted line is the emulsion boundary. Prior to illumination, GFP signal is diffuse, indicating no condensation. After 30 s of illumination and uncaging of Rap, RGG condensates appear, indicating dRap uncaging and protein dimerization. Scale bar 10 μm. (D) Kinetics measured from images of optically induced droplet formation inside emulsions, as in C. n = 10 emulsions. Shaded area, StDev.

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    Figure 4

    Figure 4. Leveraging induced dimerization to trigger synthetic condensate formation in living cells. (A) Scheme for encoding and expression of RGG polypeptides in cells, sensitive to small-molecule-induced dimerization and condensation. RGG-GFP-FKBP and RGG-FRB constructs are integrated into the yeast genome controlled by an inducible GAL1 promoter. (B) Representative images for strains described in A, showing addition of 20 μM Rap triggers condensate formation within minutes in live cells. (C) Average number of droplets formed per yeast cell in the n = 105 cells. Shaded area, 95% CI. (D) Optical regulation of condensation in cells following illumination to photouncage dRap. (E) Average number of droplets formed per cell following 10 s of 405 nm laser illumination (n = 66 cells). Shaded area, 95% CI.

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    Figure 5. Selective cargo recruitment to condensates via chemogenic dimerization. (A) Schematic of RGG-RGG condensates, which cannot selectively recruit FRB-tagged client protein (mCherry-FRB). Client does not enrich in condensates because it cannot interact with free RGG polypeptides. (Right) Representative fluorescent images of condensates from 10 μM RGG-RGG marked with 0.2 μM RGG-GFP-RGG tracer and 5 μM of client mCherry-FRB. Client is excluded from RGG-RGG condensates. (B) Scheme for cargo recruitment via rapamycin through dimerization with RGG-FKBP which partitions to condensed phase. (Right) Representative images of condensates from 10 μM each of RGG-FKBP and RGG-FRB in the presence of 10 μM Rap and 0.2 μM RGG-GFP-RGG tracer. Addition of 5 μM mCherry-FRB (client) results in robust and selective enrichment to condensed phase. (C) Kinetics of client enrichment after addition of mCherry FRB as in A and B from three independent experiments. Shaded area, 95% CI.

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  • References

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      Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates
      Alberti, Simon; Gladfelter, Amy; Mittag, Tanja
      Cell (Cambridge, MA, United States) (2019), 176 (3), 419-434CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review. Evidence is now mounting that liq.-liq. phase sepn. (LLPS) underlies the formation of membraneless compartments in cells. This realization has motivated major efforts to delineate the function of such biomol. condensates in normal cells and their roles in contexts ranging from development to age-related disease. There is great interest in understanding the underlying biophys. principles and the specific properties of biol. condensates with the goal of bringing insights into a wide range of biol. processes and systems. The explosion of physiol. and pathol. contexts involving LLPS requires clear stds. for their study. Here, we propose guidelines for rigorous exptl. characterization of LLPS processes in vitro and in cells, discuss the caveats of common exptl. approaches, and point out exptl. and theor. gaps in the field.
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    9. 9
      Peeples, W.; Rosen, M. K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. Nat. Chem. Biol. 2021, 17 (6), 693– 702,  DOI: 10.1038/s41589-021-00801-x
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      9
      Mechanistic dissection of increased enzymatic rate in a phase-separated compartment
      Peeples, William; Rosen, Michael K.
      Nature Chemical Biology (2021), 17 (6), 693-702CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
      Biomol. condensates conc. macromols. into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymic reaction rates through increased concns. of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liq.-liq. phase sepn. of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-sepn. threshold lead to a quant. model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced mol. organization. Thus, condensates can modulate reaction rates both by concg. mols. and phys. organizing them.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFKrsL3E&md5=86084e819f4718edc60b454f893426b3
    10. 10
      Zhu, L.; Richardson, T. M.; Wacheul, L.; Wei, M. T.; Feric, M.; Whitney, G.; Lafontaine, D. L. J.; Brangwynne, C. P. Controlling the material properties and rRNA processing function of the nucleolus using light. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (35), 17330– 17335,  DOI: 10.1073/pnas.1903870116
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      10
      Controlling the material properties and rRNA processing function of the nucleolus using light
      Zhu, Lian; Richardson, Tiffany M.; Wacheul, Ludivine; Wei, Ming-Tzo; Feric, Marina; Whitney, Gena; Lafontaine, Denis L. J.; Brangwynne, Clifford P.
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (35), 17330-17335CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent rRNA. A no. of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying mol. interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here the authors utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. The authors show that above a threshold concn. of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal mol. mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. The authors propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1yhurjL&md5=9517e8eae9a830967b817e6e3008842f
    11. 11
      Feric, M.; Vaidya, N.; Harmon, T. S.; Mitrea, D. M.; Zhu, L.; Richardson, T. M.; Kriwacki, R. W.; Pappu, R. V.; Brangwynne, C. P. Coexisting Liquid Phases Underlie Nucleolar Subcompartments. Cell 2016, 165 (7), 1686– 1697,  DOI: 10.1016/j.cell.2016.04.047
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      11
      Coexisting liquid phases underlie nucleolar subcompartments
      Feric, Marina; Vaidya, Nilesh; Harmon, Tyler S.; Mitrea, Diana M.; Zhu, Lian; Richardson, Tiffany M.; Kriwacki, Richard W.; Pappu, Rohit V.; Brangwynne, Clifford P.
      Cell (Cambridge, MA, United States) (2016), 165 (7), 1686-1697CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase sepn. of their mol. components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, the authors combined in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liq. phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase sep. into droplets contg. distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophys. properties of the phases, particularly droplet surface tension which arises from sequence-encoded features of their macromol. components. These results suggest that phase sepn. can give rise to multilayered liqs. that may facilitate sequential RNA processing reactions in a variety of RNP bodies.
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    12. 12
      Phair, R. D.; Misteli, T. High mobility of proteins in the mammalian cell nucleus. Nature 2000, 404 (6778), 604– 9,  DOI: 10.1038/35007077
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      12
      High mobility of proteins in the mammalian cell nucleus
      Phair, Robert D.; Misteli, Tom
      Nature (London) (2000), 404 (6778), 604-609CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The mammalian cell nucleus contains numerous sub-compartments, which have been implicated in essential processes such as transcription and splicing. The mechanisms by which nuclear compartments are formed and maintained are unclear. More fundamentally, it is not known how proteins move within the cell nucleus. We have measured the kinetic properties of proteins in the nucleus of living cells using photobleaching techniques. Here we show that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus. Protein movement is independent of energy, which indicates that proteins may use a passive mechanism of movement. Proteins rapidly assoc. and dissoc. with nuclear compartments. Using kinetic modeling, we detd. residence times and steady-state fluxes of mols. in two main nuclear compartments. These data show that many nuclear proteins roam the cell nucleus in vivo and that nuclear compartments are the reflection of the steady-state assocn./dissocn. of its 'residents' with the nucleoplasmic space. Our observations have conceptual implications for understanding nuclear architecture and how nuclear processes are organized in vivo.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXis1Grs74%253D&md5=05e4181e19d87fdbbc1faa8534926024
    13. 13
      Nott, T. J.; Petsalaki, E.; Farber, P.; Jervis, D.; Fussner, E.; Plochowietz, A.; Craggs, T. D.; Bazett-Jones, D. P.; Pawson, T.; Forman-Kay, J. D.; Baldwin, A. J. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 2015, 57 (5), 936– 947,  DOI: 10.1016/j.molcel.2015.01.013
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      13
      Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles
      Nott, Timothy J.; Petsalaki, Evangelia; Farber, Patrick; Jervis, Dylan; Fussner, Eden; Plochowietz, Anne; Craggs, Timothy D.; Bazett-Jones, David P.; Pawson, Tony; Forman-Kay, Julie D.; Baldwin, Andrew J.
      Molecular Cell (2015), 57 (5), 936-947CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Cells chem. isolate mols. in compartments to both facilitate and regulate their interactions. In addn. to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that det. when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-sepd. organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temp., ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can conc. single-stranded DNA but largely exclude double-stranded DNA. We propose that phase sepn. of disordered proteins contg. weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.
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    14. 14
      Sabari, B. R.; Dall’Agnese, A.; Boija, A.; Klein, I. A.; Coffey, E. L.; Shrinivas, K.; Abraham, B. J.; Hannett, N. M.; Zamudio, A. V.; Manteiga, J. C.; Li, C. H.; Guo, Y. E.; Day, D. S.; Schuijers, J.; Vasile, E.; Malik, S.; Hnisz, D.; Lee, T. I.; Cisse, I. I.; Roeder, R. G.; Sharp, P. A.; Chakraborty, A. K.; Young, R. A. Coactivator condensation at super-enhancers links phase separation and gene control. Science 2018, 361 (6400), eaar3958,  DOI: 10.1126/science.aar3958
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    15. 15
      Shrinivas, K.; Sabari, B. R.; Coffey, E. L.; Klein, I. A.; Boija, A.; Zamudio, A. V.; Schuijers, J.; Hannett, N. M.; Sharp, P. A.; Young, R. A.; Chakraborty, A. K. Enhancer Features that Drive Formation of Transcriptional Condensates. Mol. Cell 2019, 75 (3), 549– 561,  DOI: 10.1016/j.molcel.2019.07.009
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      15
      Enhancer Features that Drive Formation of Transcriptional Condensates
      Shrinivas, Krishna; Sabari, Benjamin R.; Coffey, Eliot L.; Klein, Isaac A.; Boija, Ann; Zamudio, Alicia V.; Schuijers, Jurian; Hannett, Nancy M.; Sharp, Phillip A.; Young, Richard A.; Chakraborty, Arup K.
      Molecular Cell (2019), 75 (3), 549-561.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-sepd. condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site no., d., and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci detd. by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase sepn. might regulate this process.
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    16. 16
      Wippich, F.; Bodenmiller, B.; Trajkovska, M. G.; Wanka, S.; Aebersold, R.; Pelkmans, L. Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 2013, 152 (4), 791– 805,  DOI: 10.1016/j.cell.2013.01.033
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      16
      Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling
      Wippich, Frank; Bodenmiller, Bernd; Trajkovska, Maria Gustafsson; Wanka, Stefanie; Aebersold, Ruedi; Pelkmans, Lucas
      Cell (Cambridge, MA, United States) (2013), 152 (4), 791-805CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Cytosolic compartmentalization through liq.-liq. unmixing, such as the formation of RNA granules, is involved in many cellular processes and might be used to regulate signal transduction. However, specific mol. mechanisms by which liq.-liq. unmixing and signal transduction are coupled remain unknown. Here, we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of P-granule-like structures and mTORC1 signaling. DYRK3 displays a cyclic partitioning mechanism between stress granules and the cytosol via a low-complexity domain in its N-terminus and its kinase activity. When DYRK3 is inactive, it prevents stress granule dissoln. and the release of sequestered mTORC1. When DYRK3 is active, it allows stress granule dissoln., releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40. This mechanism links cytoplasmic compartmentalization via liq. phase transitions with cellular signaling.
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    17. 17
      Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18 (5), 285– 298,  DOI: 10.1038/nrm.2017.7
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      17
      Biomolecular condensates: organizers of cellular biochemistry
      Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A.; Rosen, Michael K.
      Nature Reviews Molecular Cell Biology (2017), 18 (5), 285-298CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Biomol. condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to conc. proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metab., ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liq.-liq. phase sepn. driven by multivalent macromol. interactions is an important organizing principle for biomol. condensates. With this phys. framework, it is now possible to explain how the assembly, compn., phys. properties and biochem. and cellular functions of these important structures are regulated.
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    18. 18
      Brangwynne, C. P.; Tompa, P.; Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 2015, 11 (11), 899– 904,  DOI: 10.1038/nphys3532
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      Polymer physics of intracellular phase transitions
      Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.
      Nature Physics (2015), 11 (11), 899-904CODEN: NPAHAX; ISSN:1745-2473. (Nature Publishing Group)
      Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biol. roles, by compartmentalizing the cell to enable spatiotemporal control of biol. reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liq. droplets that form via phase sepn. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liq.-liq. phase sepn. in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equil. features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics.
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    19. 19
      Zumbro, E.; Alexander-Katz, A. Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation. PLoS One 2021, 16 (11), e0245405  DOI: 10.1371/journal.pone.0245405
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      19
      Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation
      Zumbro, Emiko; Alexander-Katz, Alfredo
      PLoS One (2021), 16 (11), e0245405CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Multivalent polymers are a key structural component of many biocondensates. When interacting with their cognate binding proteins, multivalent polymers such as RNA and modular proteins have been shown to influence the liq.-liq. phase sepn. (LLPS) boundary to both control condensate formation and to influence condensate dynamics after phase sepn. Much is still unknown about the function and formation of these condensed droplets, but changes in their dynamics or phase sepn. are assocd. with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's Disease. Therefore, investigation into how the structure of multivalent polymers relates to changes in biocondensate formation and maturation is essential to understanding and treating these diseases. Here, we use a coarse-grain, Brownian Dynamics simulation with reactive binding that mimics specific interactions in order to investigate the difference between non-specific and specific multivalent binding polymers. We show that non-specific binding interactions can lead to much larger changes in droplet formation at lower protein-polymer interaction energies than their specific, valence-limited counterparts. We also demonstrate the effects of solvent conditions and polymer length on phase sepn., and we present how modulating binding energy to the polymer can change the organization of a droplet in a three component system of polymer, binding protein, and solvent. Finally, we compare the effects of surface tension and polymer binding on the condensed phase dynamics, and show that both lower protein solubilities and higher attraction/affinity of the protein to the polymer result in slower droplet dynamics. This research will help to better understand exptl. systems and provides addnl. insight into how multivalent polymers can control LLPS.
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    20. 20
      Schuster, B. S.; Reed, E. H.; Parthasarathy, R.; Jahnke, C. N.; Caldwell, R. M.; Bermudez, J. G.; Ramage, H.; Good, M. C.; Hammer, D. A. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 2018, 9 (1), 2985,  DOI: 10.1038/s41467-018-05403-1
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      20
      Controllable protein phase separation and modular recruitment to form responsive membraneless organelles
      Schuster Benjamin S; Parthasarathy Ranganath; Bermudez Jessica G; Good Matthew C; Hammer Daniel A; Reed Ellen H; Jahnke Craig N; Hammer Daniel A; Caldwell Reese M; Good Matthew C; Ramage Holly
      Nature communications (2018), 9 (1), 2985 ISSN:.
      Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.
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    21. 21
      Garaizar, A.; Sanchez-Burgos, I.; Collepardo-Guevara, R.; Espinosa, J. R. Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid-Liquid Phase Separation. Molecules 2020, 25 (20), 4705,  DOI: 10.3390/molecules25204705
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      21
      Expansion of intrinsically disordered proteins increases the range of stability of liquid-liquid phase separation
      Garaizar, Adiran; Sanchez-Burgos, Ignacio; Collepardo-Guevara, Rosana; Espinosa, Jorge R.
      Molecules (2020), 25 (20), 4705CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Proteins contg. intrinsically disordered regions (IDRs) are ubiquitous within biomol. condensates, which are liq.-like compartments within cells formed through liq.-liq. phase sepn. (LLPS). The sequence of amino acids of a protein encodes its phase behavior, not only by establishing the patterning and chem. nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviors, including conformational switching, where they transition between alternate conformational ensembles. Using Mol. Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liq.-liq. phase behavior of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilizing multivalent protein-protein interactions by reducing steric hindrance; thereby, such conformations maximize the mol. connectivity of the condensed liq. network. Extended protein configurations promote phase sepn. regardless of whether LLPS is driven by homotypic and/or heterotypic protein-protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liq.-liq. phase behavior.
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    22. 22
      Flory, P. J. Principles of Polymer Chemistry; Cornell University Pres:, 1953; Vol. 1.
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      There is no corresponding record for this reference.
    23. 23
      Banani, S. F.; Rice, A. M.; Peeples, W. B.; Lin, Y.; Jain, S.; Parker, R.; Rosen, M. K. Compositional Control of Phase-Separated Cellular Bodies. Cell 2016, 166 (3), 651– 663,  DOI: 10.1016/j.cell.2016.06.010
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      23
      Compositional control of phase-separated cellular bodies
      Banani, Salman F.; Rice, Allyson M.; Peeples, William B.; Lin, Yuan; Jain, Saumya; Parker, Roy; Rosen, Michael K.
      Cell (Cambridge, MA, United States) (2016), 166 (3), 651-663CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-sepd. liqs. organized by multivalent interactions among proteins and RNA mols. Although many components of various cellular bodies are known, general principles that define body compn. are lacking. Here, the authors modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold mols. formed phase-sepd. liqs. that concd. low valency client proteins. Clients partitioned differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. The compn. could switch rapidly through changes in scaffold concn. or valency. Natural PML NBs and P bodies showed analogous partitioning behavior, suggesting how their compns. could be controlled by levels of PML SUMOylation or cellular mRNA concn., resp. The data suggested a conceptual framework for considering the compn. and control thereof of cellular bodies assembled through heterotypic multivalent interactions.
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    24. 24
      Pak, C. W.; Kosno, M.; Holehouse, A. S.; Padrick, S. B.; Mittal, A.; Ali, R.; Yunus, A. A.; Liu, D. R.; Pappu, R. V.; Rosen, M. K. Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein. Mol. Cell 2016, 63 (1), 72– 85,  DOI: 10.1016/j.molcel.2016.05.042
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      24
      Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein
      Pak, Chi W.; Kosno, Martyna; Holehouse, Alex S.; Padrick, Shae B.; Mittal, Anuradha; Ali, Rustam; Yunus, Ali A.; Liu, David R.; Pappu, Rohit V.; Rosen, Michael K.
      Molecular Cell (2016), 63 (1), 72-85CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Liq.-liq. phase sepn., driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase sepn. via complex coacervation, whereby the neg. charged NICD co-assembles with pos. charged partners to form protein-rich dense liq. droplets. Mutagenesis reveals that the driving force for phase sepn. depends on the overall amino acid compn. and not the precise sequence of NICD. Instead, phase sepn. is promoted by one or more regions of high neg. charge d. and arom./hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase sepn.
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      Quiroz, F. G.; Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 2015, 14 (11), 1164– 71,  DOI: 10.1038/nmat4418
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      25
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers
      Quiroz, Felipe Garcia; Chilkoti, Ashutosh
      Nature Materials (2015), 14 (11), 1164-1171CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)
      Proteins and synthetic polymers that undergo aq. phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behavior of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behavior, we show that the proteins can be designed to exhibit tunable lower or upper crit. soln. temp. (LCST and UCST, resp.) transitions in physiol. solns. We also show that mutation of key residues at the repeat level abolishes phase behavior or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behavior and to design novel protein polymers consisting of biol. active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behavior at the sequence level.
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    26. 26
      Zhang, H.; Elbaum-Garfinkle, S.; Langdon, E. M.; Taylor, N.; Occhipinti, P.; Bridges, A. A.; Brangwynne, C. P.; Gladfelter, A. S. RNA Controls PolyQ Protein Phase Transitions. Mol. Cell 2015, 60 (2), 220– 30,  DOI: 10.1016/j.molcel.2015.09.017
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      26
      RNA Controls PolyQ Protein Phase Transitions
      Zhang, Huaiying; Elbaum-Garfinkle, Shana; Langdon, Erin M.; Taylor, Nicole; Occhipinti, Patricia; Bridges, Andrew A.; Brangwynne, Clifford P.; Gladfelter, Amy S.
      Molecular Cell (2015), 60 (2), 220-230CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Compartmentalization in cells is central to the spatial and temporal control of biochem. In addn. to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liq.-liq. phase sepn. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and phys. identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiol. targets of Whi3 drive phase sepn. MRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk soln. Different mRNAs impart distinct biophys. properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophys. properties of phase-sepd. compartments.
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    27. 27
      Lytle, T. K.; Sing, C. E. Tuning chain interaction entropy in complex coacervation using polymer stiffness, architecture, and salt valency. Molecular Systems Design & Engineering 2018, 3 (1), 183– 196,  DOI: 10.1039/C7ME00108H
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      Tuning chain interaction entropy in complex coacervation using polymer stiffness, architecture, and salt valency
      Lytle, Tyler K.; Sing, Charles E.
      Molecular Systems Design & Engineering (2018), 3 (1), 183-196CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
      Oppositely-charged polyelectrolytes can undergo a liq.-liq. phase sepn. in a salt soln., resulting in a polymer-dense 'coacervate' phase that has found use in a wide range of applications from food science to self-assembled materials. Coacervates can be tuned for specific applications by varying parameters such as salt concn. and valency, polyelectrolyte length, and polyelectrolyte identity. Recent simulation and theory has begun to clarify the role of mol. structure on coacervation phase behavior, esp. for common synthetic polyelectrolytes that exhibit high charge densities. In this manuscript, we use a combination of transfer matrix theory and Monte Carlo simulation to understand at a phys. level how a range of mol. features, in particular polymer architecture and stiffness, and salt valency can be used to design the phase diagrams of these materials. We demonstrate a phys. picture of how the underlying entropy-driven process of complex coacervation is affected by this wide range of phys. attributes.
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    28. 28
      Garaizar, A.; Espinosa, J. R.; Joseph, J. A.; Collepardo-Guevara, R. Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates. Sci. Rep 2022, 12 (1), 4390,  DOI: 10.1038/s41598-022-08130-2
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      28
      Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates
      Garaizar, Adiran; Espinosa, Jorge R.; Joseph, Jerelle A.; Collepardo-Guevara, Rosana
      Scientific Reports (2022), 12 (1), 4390CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
      Biomol. condensates formed by the process of liq.-liq. phase sepn. (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chem. reactions. Upon maturation, the liq.-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathol. implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a mol.-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodn. properties of biomol. condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equil. condensate has been formed, or when they accumulate slowly over time with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides-prone to forming inter-protein β-sheets-we observe that formation of inter-peptide β-sheets increases the strength of the interactions consistently with the loss of liq.-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental mol., kinetic, and thermodn. mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing.
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    29. 29
      Garabedian, M. V.; Wang, W.; Dabdoub, J. B.; Tong, M.; Caldwell, R. M.; Benman, W.; Schuster, B. S.; Deiters, A.; Good, M. C. Designer membraneless organelles sequester native factors for control of cell behavior. Nat. Chem. Biol. 2021, 17 (9), 998– 1007,  DOI: 10.1038/s41589-021-00840-4
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      29
      Designer membraneless organelles sequester native factors for control of cell behavior
      Garabedian, Mikael V.; Wang, Wentao; Dabdoub, Jorge B.; Tong, Michelle; Caldwell, Reese M.; Benman, William; Schuster, Benjamin S.; Deiters, Alexander; Good, Matthew C.
      Nature Chemical Biology (2021), 17 (9), 998-1007CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
      Subcellular compartmentalization of macromols. increases flux and prevents inhibitory interactions to control biochem. reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.
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    30. 30
      Perdikari, T. M.; Jovic, N.; Dignon, G. L.; Kim, Y. C.; Fawzi, N. L.; Mittal, J. A predictive coarse-grained model for position-specific effects of post-translational modifications. Biophys. J. 2021, 120 (7), 1187– 1197,  DOI: 10.1016/j.bpj.2021.01.034
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      A predictive coarse-grained model for position-specific effects of post-translational
      Perdikari, Theodora Myrto; Jovic, Nina; Dignon, Gregory L.; Kim, Young C.; Fawzi, Nicolas L.; Mittal, Jeetain
      Biophysical Journal (2021), 120 (7), 1187-1197CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Biomols. undergo liq.-liq. phase sepn. (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiol. and pathol. role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the no. and position of phosphorylated and acetylated residues alter the protein's single-chain compactness and its propensity to phase sep. We show that both the no. and the position of phosphorylated threonines/serines or acetylated lysines can serve as a mol. on/off switch for phase sepn. in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, resp. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture exptl. measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase sepn. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resoln.
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      Owen, I.; Shewmaker, F. The Role of Post-Translational Modifications in the Phase Transitions of Intrinsically Disordered Proteins. Int. J. Mol. Sci. 2019, 20 (21), 5501,  DOI: 10.3390/ijms20215501
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      The role of post-translational modifications in the phase transitions of intrinsically disordered proteins
      Owen, Izzy; Shewmaker, Frank
      International Journal of Molecular Sciences (2019), 20 (21), 5501CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. Advances in genomics and proteomics have revealed eukaryotic proteomes to be highly abundant in intrinsically disordered proteins that are susceptible to diverse post-translational modifications. Intrinsically disordered regions are crit. to the liq.-liq. phase sepn. that facilitates specialized cellular functions. Here, we discuss how post-translational modifications of intrinsically disordered protein segments can regulate the mol. condensation of macromols. into functional phase-sepd. complexes.
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      Söding, J.; Zwicker, D.; Sohrabi-Jahromi, S.; Boehning, M.; Kirschbaum, J. Mechanisms for Active Regulation of Biomolecular Condensates. Trends Cell Biol. 2020, 30 (1), 4– 14,  DOI: 10.1016/j.tcb.2019.10.006
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      Mechanisms for Active Regulation of Biomolecular Condensates
      Soding Johannes; Zwicker David; Kirschbaum Jan; Sohrabi-Jahromi Salma; Boehning Marc
      Trends in cell biology (2020), 30 (1), 4-14 ISSN:.
      Liquid-liquid phase separation is a key organizational principle in eukaryotic cells, on par with intracellular membranes. It allows cells to concentrate specific proteins into condensates, increasing reaction rates and achieving switch-like regulation. We propose two active mechanisms that can explain how cells regulate condensate formation and size. In both, the cell regulates the activity of an enzyme, often a kinase, that adds post-translational modifications to condensate proteins. In enrichment inhibition, the enzyme enriches in the condensate and weakens interactions, as seen in stress granules (SGs), Cajal bodies, and P granules. In localization-induction, condensates form around immobilized enzymes that strengthen interactions, as observed in DNA repair, transmembrane signaling, and microtubule assembly. These models can guide studies into the many emerging roles of biomolecular condensates.
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      Song, D.; Jo, Y.; Choi, J. M.; Jung, Y. Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions. Nat. Commun. 2020, 11 (1), 5642,  DOI: 10.1038/s41467-020-19476-4
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      Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions
      Song, Daesun; Jo, Yongsang; Choi, Jeong-Mo; Jung, Yongwon
      Nature Communications (2020), 11 (1), 5642CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liq.-liq. phase sepn., biomol. behaviors such as protein interactions inside these liq. compartments have been largely unexplored. Here we report quant. measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase sepn. conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase sepn. model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concns. inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase sepn. compartmentalization: (1) clients are selectively recruited into compartments, leading to concn. enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.
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      Zhang, H.; Zhao, R.; Tones, J.; Liu, M.; Dilley, R. L.; Chenoweth, D. M.; Greenberg, R. A.; Lampson, M. A. Nuclear body phase separation drives telomere clustering in ALT cancer cells. Mol. Biol. Cell 2020, 31 (18), 2048– 2056,  DOI: 10.1091/mbc.E19-10-0589
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      Nuclear body phase separation drives telomere clustering in ALT cancer cells
      Zhang, Huaiying; Zhao, Rongwei; Tones, Jason; Liu, Michel; Dilley, Robert L.; Chenoweth, David M.; Greenberg, Roger A.; Lampson, Michael A.
      Molecular Biology of the Cell (2020), 31 (18), 2048-2056CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      A review. Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-assocd. promyelocytic leukemia nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liq. condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chem. induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere-binding protein sumoylation nucleates APB condensation via interactions between small ubiquitin-like modifier (SUMO) and SUMO interaction motif (SIM), and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liq. properties of the condensate, as an alternative condensation chem. also induces clustering independent of sumoylation. Our findings introduce a chem. dimerization approach to manipulate phase sepn. and demonstrate how the material properties and chem. compn. of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions.
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      Wang, A.; Conicella, A. E.; Schmidt, H. B.; Martin, E. W.; Rhoads, S. N.; Reeb, A. N.; Nourse, A.; Ramirez Montero, D.; Ryan, V. H.; Rohatgi, R.; Shewmaker, F.; Naik, M. T.; Mittag, T.; Ayala, Y. M.; Fawzi, N. L. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. Embo j 2018, 37 (5), e97452  DOI: 10.15252/embj.201797452
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      Li, P.; Banjade, S.; Cheng, H. C.; Kim, S.; Chen, B.; Guo, L.; Llaguno, M.; Hollingsworth, J. V.; King, D. S.; Banani, S. F.; Russo, P. S.; Jiang, Q. X.; Nixon, B. T.; Rosen, M. K. Phase transitions in the assembly of multivalent signalling proteins. Nature 2012, 483 (7389), 336– 40,  DOI: 10.1038/nature10879
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      Phase transitions in the assembly of multivalent signalling proteins
      Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V.; King, David S.; Banani, Salman F.; Russo, Paul S.; Jiang, Qiu-Xing; Nixon, B. Tracy; Rosen, Michael K.
      Nature (London, United Kingdom) (2012), 483 (7389), 336-340CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Cells are organized on length scales ranging from angstroms to micrometers. However, the mechanisms by which angstrom-scale mol. properties are translated to micrometer-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromols. (including multidomain proteins and RNA) produce sharp liq.-liq.-demixing phase sepns., generating micrometer-sized liq. droplets in aq. soln. This macroscopic transition corresponds to a mol. transition between small complexes and large, dynamic supramol. polymers. The concns. needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biol. partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochem. regulate information throughout biol.
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      Roden, C.; Gladfelter, A. S. RNA contributions to the form and function of biomolecular condensates. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 183– 195,  DOI: 10.1038/s41580-020-0264-6
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      RNA contributions to the form and function of biomolecular condensates
      Roden, Christine; Gladfelter, Amy S.
      Nature Reviews Molecular Cell Biology (2021), 22 (3), 183-195CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
      A review. Biomol. condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liq.-liq. phase sepn. (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomol. condensation and highlight considerations for designing condensate reconstitution expts. We focus on RNA properties such as compn., length, structure, modifications and expression level. These properties can modulate the biophys. features of native condensates, including their size, shape, viscosity, liquidity, surface tension and compn. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be detd. by RNA. Finally, we discuss the multifaceted cellular functions of biomol. condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific mols., and buffering noise and responding to stress.
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      Tejedor, A. R.; Garaizar, A.; Ramírez, J.; Espinosa, J. R. RNA modulation of transport properties and stability in phase-separated condensates. Biophys. J. 2021, 120 (23), 5169– 5186,  DOI: 10.1016/j.bpj.2021.11.003
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      'RNA modulation of transport properties and stability in phase-separated condensates
      Tejedor, Andres R.; Garaizar, Adiran; Ramirez, Jorge; Espinosa, Jorge R.
      Biophysical Journal (2021), 120 (23), 5169-5186CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissoln. of phase-sepd. condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chem.-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomol. condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concn. By characterizing the phase diagram, key mol. interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase sepn. at low concn. as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concn., it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concn. as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concn., but barely modify protein self-diffusion which mainly depends on RNA concn. and on the effect RNA has on droplet d. On the whole, our work rationalizes the different routes by which RNA can regulate phase sepn. and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.
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    39. 39
      Li, N. K.; Roberts, S.; Quiroz, F. G.; Chilkoti, A.; Yingling, Y. G. Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides. Biomacromolecules 2018, 19 (7), 2496– 2505,  DOI: 10.1021/acs.biomac.8b00099
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      Sequence directionality dramatically affects LCST behavior of elastin-like polypeptides
      Li, Nan K.; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G.
      Biomacromolecules (2018), 19 (7), 2496-2505CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Elastin-like polypeptides (ELP) exhibit an inverse temp. transition or lower crit. soln. temp. (LCST) transition phase behavior in aq. solns. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, CD (CD) measurements, and all-atom mol. dynamics (MD) simulations to gain a mol. understanding of mechanism that controls hysteretic phase behavior. It was shown exptl. that both poly(VPGVG) and poly(VGPVG) undergo a transition from sol. to insol. in aq. soln. upon heating above the transition temp. (Tt). However, poly(VPGVG) resolubilizes upon cooling below its Tt, whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the Tt. The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, esp. valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above Tt, where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the obsd. thermal hysteresis for poly(VGPVG).
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      Dzuricky, M.; Rogers, B. A.; Shahid, A.; Cremer, P. S.; Chilkoti, A. De novo engineering of intracellular condensates using artificial disordered proteins. Nat. Chem. 2020, 12 (9), 814– 825,  DOI: 10.1038/s41557-020-0511-7
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      De novo engineering of intracellular condensates using artificial disordered proteins
      Dzuricky, Michael; Rogers, Bradley A.; Shahid, Abdulla; Cremer, Paul S.; Chilkoti, Ashutosh
      Nature Chemistry (2020), 12 (9), 814-825CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      Phase sepn. of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, the authors systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different mol. wts. and arom. content, which exhibit variable condensate satn. concns. and temp. cloud points in vitro and in cells. The authors created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the mol. wt. of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-sepn.-mediated control of biol. function in living cells.
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    41. 41
      Basheer, A.; Shahid, S.; Kang, M. J.; Lee, J. H.; Lee, J. S.; Lim, D. W. Switchable Self-Assembly of Elastin- and Resilin-Based Block Copolypeptides with Converse Phase Transition Behaviors. ACS Appl. Mater. Interfaces 2021, 13 (21), 24385– 24400,  DOI: 10.1021/acsami.1c00676
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      41
      Switchable Self-Assembly of Elastin- and Resilin-Based Block Copolypeptides with Converse Phase Transition Behaviors
      Basheer, Aamna; Shahid, Shahzaib; Kang, Min Jung; Lee, Jae Hee; Lee, Jae Sang; Lim, Dong Woo
      ACS Applied Materials & Interfaces (2021), 13 (21), 24385-24400CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Self-assembly of thermally responsive polypeptides into unique nanostructures offers intriguing attributes including dynamic phys. dimensions, biocompatibility, and biodegradability for the smart bio-nanomaterials. As elastin-based polypeptide (EBP) fusion proteins with lower crit. soln. temp. (LCST) are studied as drug delivery systems, EBP block copolypeptides with the resilin-based polypeptide (RBP) displaying an upper crit. soln. temp. (UCST) have been of great interest. In this study, we report thermally triggered, dynamic self-assembly of EBP- and RBP-based diblock copolypeptides into switched nanostructures with reversibility under physiol. conditions. Mol. DNA clones encoding for the EBP-RBP diblocks at different block length ratios were biosynthesized via recursive directional ligation and overexpressed, followed by nonchromatog. purifn. by inverse transition cycling. Genetically engineered diblock copolypeptides composed of the EBP with an LCST and the RBP with a UCST showed converse phase transition behaviors with both a distinct LCST and a distinct UCST (LCST < UCST). As temp. increased, three phases of these EBP-RBP diblocks were obsd.: (1) self-assembled micelles or vesicles below both LCST and UCST, (2) whole aggregates above LCST and below UCST, and (3) reversed micelles above both LCST and UCST. In conclusion, these stimuli-triggered, dynamic protein-based nanostructures are promising for advanced drug delivery systems, regenerative medicine, and biomedical nanotechnol.
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    42. 42
      Reed, E. H.; Hammer, D. A. Redox sensitive protein droplets from recombinant oleosin. Soft Matter 2018, 14 (31), 6506– 6513,  DOI: 10.1039/C8SM01047A
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      42
      Redox sensitive protein droplets from recombinant oleosin
      Reed, Ellen H.; Hammer, Daniel A.
      Soft Matter (2018), 14 (31), 6506-6513CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
      Protein engineering enables the creation of materials with designer functionality and tailored responsiveness. Here, we design a protein with two control motifs for its phase sepn. into micron sized liq. droplets - one driven by a hydrophobic domain and the other by oxidn. of a disulfide bond. Our work is based on the plant surfactant protein, oleosin, which has a hydrophobic domain but no cysteines. Oleosin phase separates to form liq. droplets below a crit. temp. akin to many naturally occurring membrane-less organelles. Sequence mutations are made to introduce a cysteine residue into oleosin. The addn. of a cysteine causes phase sepn. at a lower concn. and increases the phase transition temp. Adding a reducing agent to phase-sepd., cysteine-contg. oleosin rapidly dissolves the droplets. The transition temp. is tuned by varying the location of the cysteine or by blending the parent cysteine-less mol. with the cysteine-contg. mutant. This provides a novel way to control protein droplet formation and dissoln. We envision this work having applications as a system for the release of a protein or drug with engineered sensitivity to reducing conditions and as a mimic of membrane-less organelles in synthetic protocells.
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    43. 43
      Costa, S. A.; Simon, J. R.; Amiram, M.; Tang, L.; Zauscher, S.; Brustad, E. M.; Isaacs, F. J.; Chilkoti, A. Photo-Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano- to Microgels of Intrinsically Disordered Polypeptides. Adv. Mater. 2018, 30 (5), 1704878,  DOI: 10.1002/adma.201704878
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      Agouridas, V.; El Mahdi, O.; Diemer, V.; Cargoët, M.; Monbaliu, J. M.; Melnyk, O. Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations. Chem. Rev. 2019, 119 (12), 7328– 7443,  DOI: 10.1021/acs.chemrev.8b00712
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      44
      Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations
      Agouridas, Vangelis; El Mahdi, Ouafaa; Diemer, Vincent; Cargoet, Marine; Monbaliu, Jean-Christophe M.; Melnyk, Oleg
      Chemical Reviews (Washington, DC, United States) (2019), 119 (12), 7328-7443CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The native chem. ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnol. research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and phys. chem. studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O,S-, N,S-, or N,Se-acyl shift systems as thioester or selenoester surrogates and discusses the special mol. features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N,S-acyl migrations and selective dechalcogenation reactions.
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    45. 45
      Keeble, A. H.; Howarth, M. Power to the protein: enhancing and combining activities using the Spy toolbox. Chem. Sci. 2020, 11 (28), 7281– 7291,  DOI: 10.1039/D0SC01878C
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      Power to the protein: enhancing and combining activities using the Spy toolbox
      Keeble, Anthony H.; Howarth, Mark
      Chemical Science (2020), 11 (28), 7281-7291CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A review. Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein anal. or application. Here we review SpyTag technol., now used in hundreds of publications or patents, and its potential for detecting and controlling protein behavior. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purifn. of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. SpyTag/SpyCatcher is mech. stable, so is widely used for investigating protein folding and force sensitivity. A toolbox of scaffolds allows SpyTag-fusions to be assembled into defined multimers, from dimers to 180-mers, or unlimited 1D, 2D or 3D networks. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. For enzymes, Spy technol. has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors.
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    46. 46
      Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. Characterization of the FKBP.rapamycin.FRB ternary complex. J. Am. Chem. Soc. 2005, 127 (13), 4715– 21,  DOI: 10.1021/ja043277y
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      46
      Characterization of the FKBP·Rapamycin·FRB Ternary Complex
      Banaszynski, Laura A.; Liu, Corey W.; Wandless, Thomas J.
      Journal of the American Chemical Society (2005), 127 (13), 4715-4721CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough anal. of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP·rapamycin·FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Anal. of the data shows that rapamycin binds to FRB with moderate affinity (Kd = 26±0.8 μM). The FKBP12·rapamycin complex, however, binds to FRB 2000-fold more tightly (Kd = 12±0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiol. conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.
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    47. 47
      Caldwell, R. M.; Bermudez, J. G.; Thai, D.; Aonbangkhen, C.; Schuster, B. S.; Courtney, T.; Deiters, A.; Hammer, D. A.; Chenoweth, D. M.; Good, M. C. Optochemical Control of Protein Localization and Activity within Cell-like Compartments. Biochemistry 2018, 57 (18), 2590– 2596,  DOI: 10.1021/acs.biochem.8b00131
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      47
      Optochemical control of protein localization and activity within cell-like compartments
      Caldwell, Reese M.; Bermudez, Jessica G.; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S.; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A.; Chenoweth, David M.; Good, Matthew C.
      Biochemistry (2018), 57 (18), 2590-2596CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      We report inducible dimerization strategies for controlling protein positioning, enzymic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compd., we demonstrate small mol. and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biol. processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a crit. step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
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    48. 48
      Ballister, E. R.; Aonbangkhen, C.; Mayo, A. M.; Lampson, M. A.; Chenoweth, D. M. Localized light-induced protein dimerization in living cells using a photocaged dimerizer. Nat. Commun. 2014, 5, 5475,  DOI: 10.1038/ncomms6475
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      48
      Localized light-induced protein dimerization in living cells using a photocaged dimerizer
      Ballister Edward R; Mayo Alyssa M; Lampson Michael A; Aonbangkhen Chanat; Chenoweth David M
      Nature communications (2014), 5 (), 5475 ISSN:.
      Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein-protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.
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    49. 49
      Inobe, T.; Nukina, N. Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins. J. Biosci Bioeng 2016, 122 (1), 40– 6,  DOI: 10.1016/j.jbiosc.2015.12.004
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      49
      Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins
      Inobe, Tomonao; Nukina, Nobuyuki
      Journal of Bioscience and Bioengineering (2016), 122 (1), 40-46CODEN: JBBIF6; ISSN:1347-4421. (Society for Biotechnology, Japan)
      Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addn. of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biol. applications.
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    50. 50
      Reed, E. H.; Schuster, B. S.; Good, M. C.; Hammer, D. A. SPLIT: Stable Protein Coacervation Using a Light Induced Transition. ACS Synth. Biol. 2020, 9 (3), 500– 507,  DOI: 10.1021/acssynbio.9b00503
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      50
      SPLIT: Stable Protein Coacervation Using a Light Induced Transition
      Reed, Ellen H.; Schuster, Benjamin S.; Good, Matthew C.; Hammer, Daniel A.
      ACS Synthetic Biology (2020), 9 (3), 500-507CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Protein coacervates serve as hubs to conc. and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiol. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein contg. a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase sepn. using light.
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    51. 51
      Schuster, B. S.; Dignon, G. L.; Tang, W. S.; Kelley, F. M.; Ranganath, A. K.; Jahnke, C. N.; Simpkins, A. G.; Regy, R. M.; Hammer, D. A.; Good, M. C.; Mittal, J. Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (21), 11421– 11431,  DOI: 10.1073/pnas.2000223117
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      51
      Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior
      Schuster, Benjamin S.; Dignon, Gregory L.; Tang, Wai Shing; Kelley, Fleurie M.; Ranganath, Aishwarya Kanchi; Jahnke, Craig N.; Simpkins, Alison G.; Regy, Roshan Mammen; Hammer, Daniel A.; Good, Matthew C.; Mittal, Jeetain
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (21), 11421-11431CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Phase sepn. of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize mols. intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase sepn. is crit. for understanding physiol. roles and pathol. consequences of biomol. condensation, as well as for harnessing phase sepn. for applications in bioinspired materials design. To expand the authors' knowledge of sequence determinants of IDP phase sepn., the authors characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase sepn. and a key component of P granules. Based on a predictive coarse-grained IDP model, the authors identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase sepn. in vitro and in vivo. The authors detd. the effects of charge patterning on phase behavior through sequence shuffling. The authors designed sequences with significantly increased phase sepn. propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under neg. selection to moderate this mode of interaction. The authors measured the contributions of tyrosine and arginine residues to phase sepn. exptl. through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, despite these sequence perturbations, the RGG-derived condensates remain liq.-like. Together, these studies advance the authors' fundamental understanding of key biophys. principles and sequence features important to phase sepn.
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    52. 52
      Apostolovic, B.; Danial, M.; Klok, H. A. Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem. Soc. Rev. 2010, 39 (9), 3541– 75,  DOI: 10.1039/b914339b
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      52
      Coiled coils: Attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials
      Apostolovic, Bojana; Danial, Maarten; Klok, Harm-Anton
      Chemical Society Reviews (2010), 39 (9), 3541-3575CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. The coiled-coil is a superhelical protein structural motif that consists of 2 or more α-helical peptides that are wrapped around each other in superhelical fashion. Coiled-coils are among the most ubiquitous folding motifs found in proteins and have not only been identified in structural proteins but also play an important role in various intracellular regulation processes as well as membrane fusion. The aim of this crit. review is to highlight the potential of coiled coil peptide sequences for the development of self-assembled, responsive and/or bioactive materials. After a short historical overview outlining the discovery of this protein folding motif, the article briefly discusses naturally occurring coiled-coils. After that, the basic rules, which have been established to date for the design of coiled-coils is briefly summarized followed by a presentation of several classes of coiled-coils, which may represent interesting candidates for the development of novel self-assembled, responsive and/or bioactive materials. This crit. review ends with a section that summarizes the different coiled-coil-based (hybrid) materials that have been reported to date and which hopefully will help to stimulate further work to explore the full potential of this unique class of protein folding motifs for the development of novel self-assembled, responsive and/or bioactive materials.
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    53. 53
      Fletcher, J. M.; Boyle, A. L.; Bruning, M.; Bartlett, G. J.; Vincent, T. L.; Zaccai, N. R.; Armstrong, C. T.; Bromley, E. H.; Booth, P. J.; Brady, R. L.; Thomson, A. R.; Woolfson, D. N. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology. ACS Synth. Biol. 2012, 1 (6), 240– 50,  DOI: 10.1021/sb300028q
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      53
      A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology
      Fletcher, Jordan M.; Boyle, Aimee L.; Bruning, Marc; Bartlett, Gail J.; Vincent, Thomas L.; Zaccai, Nathan R.; Armstrong, Craig T.; Bromley, Elizabeth H. C.; Booth, Paula J.; Brady, R. Leo; Thomson, Andrew R.; Woolfson, Derek N.
      ACS Synthetic Biology (2012), 1 (6), 240-250CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Protein engineering, chem. biol., and synthetic biol. would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and anal. of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in soln. by circular-dichroism spectroscopy and anal. ultracentrifugation, and their structures were detd. by X-ray crystallog. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed exptl. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chem. and synthetic biol. To help with such endeavors, we introduce Pcomp, an online registry of peptide components for protein-design and synthetic-biol. applications.
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    54. 54
      Majerle, A.; Hadži, S.; Aupič, J.; Satler, T.; Lapenta, F.; Strmšek, Ž.; Lah, J.; Loris, R.; Jerala, R. A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (17), e2021899118  DOI: 10.1073/pnas.2021899118
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      54
      A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures
      Majerle, Andreja; Hadzi, San; Aupic, Jana; Satler, Tadej; Lapenta, Fabio; Strmsek, Ziga; Lah, Jurij; Loris, Remy; Jerala, Roman
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (17), e2021899118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that det. the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong pos. cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.
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    55. 55
      Georgoulia, P. S.; Bjelic, S. Prediction of Protein-Protein Binding Interactions in Dimeric Coiled Coils by Information Contained in Folding Energy Landscapes. Int. J. Mol. Sci. 2021, 22 (3), 1368,  DOI: 10.3390/ijms22031368
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      55
      Prediction of protein-protein binding interactions in dimeric coiled coils by information contained in folding energy landscapes
      Georgoulia, Panagiota S.; Bjelic, Sinisa
      International Journal of Molecular Sciences (2021), 22 (3), 1368CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      Coiled coils represent the simplest form of a complex formed between two interacting protein partners. Their extensive study has led to the development of various methods aimed towards the investigation and design of complex forming interactions. Despite the progress that has been made to predict the binding affinities for protein complexes, and specifically those tailored towards coiled coils, many challenges still remain. In this work, we explore whether the information contained in dimeric coiled coil folding energy landscapes can be used to predict binding interactions. Using the published SYNZIP dataset, we start from the amino acid sequence, to simultaneously fold and dock approx. 1000 coiled coil dimers. Assessment of the folding energy landscapes showed that a model based on the calcd. no. of clusters for the lowest energy structures displayed a signal that correlates with the exptl. detd. protein interactions. Although the revealed correlation is weak, we show that such correlation exists; however, more work remains to establish whether further improvements can be made to the presented model.
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    56. 56
      Thompson, K. E.; Bashor, C. J.; Lim, W. A.; Keating, A. E. SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth. Biol. 2012, 1 (4), 118– 29,  DOI: 10.1021/sb200015u
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      56
      SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo Specifications of Heterospecific Coiled-Coil Interaction Domains
      Thompson, Kenneth Evan; Bashor, Caleb J.; Lim, Wendell A.; Keating, Amy E.
      ACS Synthetic Biology (2012), 1 (4), 118-129CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The synthetic biol. toolkit contains a growing no. of parts for regulating transcription and translation, but very few that can be used to control protein assocn. Here the authors report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. The authors present biophys. anal. of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the no. of available protein interaction parts for synthetic biol. and should facilitate a wide range of mol. engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].
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    57. 57
      Lebar, T.; Lainšček, D.; Merljak, E.; Aupič, J.; Jerala, R. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat. Chem. Biol. 2020, 16 (5), 513– 519,  DOI: 10.1038/s41589-019-0443-y
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      A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells
      Lebar, Tina; Lainscek, Dusko; Merljak, Estera; Aupic, Jana; Jerala, Roman
      Nature Chemical Biology (2020), 16 (5), 513-519CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biol. systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small mol.-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biol.
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    58. 58
      Shin, Y.; Berry, J.; Pannucci, N.; Haataja, M. P.; Toettcher, J. E.; Brangwynne, C. P. Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Cell 2017, 168 (1–2), 159– 171,  DOI: 10.1016/j.cell.2016.11.054
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      Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets
      Shin, Yongdae; Berry, Joel; Pannucci, Nicole; Haataja, Mikko P.; Toettcher, Jared E.; Brangwynne, Clifford P.
      Cell (Cambridge, MA, United States) (2017), 168 (1-2), 159-171.e14CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liq.-like ribonucleoprotein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiol. and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concn. threshold, these constructs undergo light-activated phase sepn., forming spatiotemporally definable liq. optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiol. phase transitions but also their link to pathol. aggregates.
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    59. 59
      Bracha, D.; Walls, M. T.; Wei, M. T.; Zhu, L.; Kurian, M.; Avalos, J. L.; Toettcher, J. E.; Brangwynne, C. P. Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds. Cell 2018, 175 (6), 1467– 1480,  DOI: 10.1016/j.cell.2018.10.048
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      Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds
      Bracha, Dan; Walls, Mackenzie T.; Wei, Ming-Tzo; Zhu, Lian; Kurian, Martin; Avalos, Jose L.; Toettcher, Jared E.; Brangwynne, Clifford P.
      Cell (Cambridge, MA, United States) (2018), 175 (6), 1467-1480.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Liq.-liq. phase sepn. plays a key role in the assembly of diverse intracellular structures. However, the biophys. principles by which phase sepn. can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quant. light-controlled tunability to map full intracellular phase diagrams, which dictate the concns. at which phase sepn. occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both expts. and simulations show that while intracellular concns. may be insufficient for global phase sepn., sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase sepn. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates assocd. with diverse biol. processes.
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    60. 60
      Wei, S. P.; Qian, Z. G.; Hu, C. F.; Pan, F.; Chen, M. T.; Lee, S. Y.; Xia, X. X. Formation and functionalization of membraneless compartments in Escherichia coli. Nat. Chem. Biol. 2020, 16 (10), 1143– 1148,  DOI: 10.1038/s41589-020-0579-9
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      Formation and functionalization of membraneless compartments in Escherichia coli
      Wei, Shao-Peng; Qian, Zhi-Gang; Hu, Chun-Fei; Pan, Fang; Chen, Meng-Ting; Lee, Sang Yup; Xia, Xiao-Xia
      Nature Chemical Biology (2020), 16 (10), 1143-1148CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Membraneless organelles formed by liq.-liq. phase sepn. of proteins or nucleic acids are involved in diverse biol. processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liq. protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiol. crowding environments of E. coli revealed that the condensates are formed via liq.-liq. phase sepn. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochem. reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biol.
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      Zhao, E. M.; Suek, N.; Wilson, M. Z.; Dine, E.; Pannucci, N. L.; Gitai, Z.; Avalos, J. L.; Toettcher, J. E. Light-based control of metabolic flux through assembly of synthetic organelles. Nat. Chem. Biol. 2019, 15 (6), 589– 597,  DOI: 10.1038/s41589-019-0284-8
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      Light-based control of metabolic flux through assembly of synthetic organelles
      Zhao, Evan M.; Suek, Nathan; Wilson, Maxwell Z.; Dine, Elliot; Pannucci, Nicole L.; Gitai, Zemer; Avalos, Jose L.; Toettcher, Jared E.
      Nature Chemical Biology (2019), 15 (6), 589-597CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      To maximize a desired product, metabolic engineers typically express enzymes to high, const. levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabs. in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concn. of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
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    62. 62
      Aonbangkhen, C.; Zhang, H.; Wu, D. Z.; Lampson, M. A.; Chenoweth, D. M. Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer. J. Am. Chem. Soc. 2018, 140 (38), 11926– 11930,  DOI: 10.1021/jacs.8b07753
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      62
      Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer
      Aonbangkhen, Chanat; Zhang, Huaiying; Wu, Daniel Z.; Lampson, Michael A.; Chenoweth, David M.
      Journal of the American Chemical Society (2018), 140 (38), 11926-11930CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Many dynamic biol. processes are regulated by protein-protein interactions and protein localization. Exptl. techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chem. induced dimerization (CID) of proteins. CID has been used to study several cellular events, esp. cell signaling networks, which are often reversible. However, chem. dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resoln. are currently limited. Herein, we present a novel chem. inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this mol. by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biol. questions on subcellular levels by controlling protein-protein interactions.
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      Haruki, H.; Nishikawa, J.; Laemmli, U. K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 2008, 31 (6), 925– 32,  DOI: 10.1016/j.molcel.2008.07.020
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      The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes
      Haruki, Hirohito; Nishikawa, Junichi; Laemmli, Ulrich K.
      Molecular Cell (2008), 31 (6), 925-932CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
      The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addn. of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addn., when examd. functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biol. to dissect the function of individual or gene pairs in synthetic, lethal situations.
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      Woods, B.; Kuo, C. C.; Wu, C. F.; Zyla, T. R.; Lew, D. J. Polarity establishment requires localized activation of Cdc42. J. Cell Biol. 2015, 211 (1), 19– 26,  DOI: 10.1083/jcb.201506108
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      Polarity establishment requires localized activation of Cdc42
      Woods, Benjamin; Kuo, Chun-Chen; Wu, Chi-Fang; Zyla, Trevin R.; Lew, Daniel J.
      Journal of Cell Biology (2015), 211 (1), 19-26CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the front of the cell. The high local concn. of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve pos. feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct pos. feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system.
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      Langan, R. A.; Boyken, S. E.; Ng, A. H.; Samson, J. A.; Dods, G.; Westbrook, A. M.; Nguyen, T. H.; Lajoie, M. J.; Chen, Z.; Berger, S.; Mulligan, V. K.; Dueber, J. E.; Novak, W. R. P.; El-Samad, H.; Baker, D. De novo design of bioactive protein switches. Nature 2019, 572 (7768), 205– 210,  DOI: 10.1038/s41586-019-1432-8
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      De novo design of bioactive protein switches
      Langan, Robert A.; Boyken, Scott E.; Ng, Andrew H.; Samson, Jennifer A.; Dods, Galen; Westbrook, Alexandra M.; Nguyen, Taylor H.; Lajoie, Marc J.; Chen, Zibo; Berger, Stephanie; Mulligan, Vikram Khipple; Dueber, John E.; Novak, Walter R. P.; El-Samad, Hana; Baker, David
      Nature (London, United Kingdom) (2019), 572 (7768), 205-210CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Allosteric regulation of protein function is widespread in biol., but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramol. interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degrdn. or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biol. and cell engineering.
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    68. 68
      Ng, A. H.; Nguyen, T. H.; Gómez-Schiavon, M.; Dods, G.; Langan, R. A.; Boyken, S. E.; Samson, J. A.; Waldburger, L. M.; Dueber, J. E.; Baker, D.; El-Samad, H. Modular and tunable biological feedback control using a de novo protein switch. Nature 2019, 572 (7768), 265– 269,  DOI: 10.1038/s41586-019-1425-7
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      Modular and tunable biological feedback control using a de novo protein switch
      Ng, Andrew H.; Nguyen, Taylor H.; Gomez-Schiavon, Mariana; Dods, Galen; Langan, Robert A.; Boyken, Scott E.; Samson, Jennifer A.; Waldburger, Lucas M.; Dueber, John E.; Baker, David; El-Samad, Hana
      Nature (London, United Kingdom) (2019), 572 (7768), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technol.8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here the authors leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signaling pathways and synthetic gene circuits. The authors first generate synthetic neg. and pos. feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signaling mols., illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. The authors next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behavior in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biol.10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnol. and therapeutic applications.
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    69. 69
      Ljubetič, A.; Lapenta, F.; Gradišar, H.; Drobnak, I.; Aupič, J.; Strmšek, Ž.; Lainšček, D.; Hafner-Bratkovič, I.; Majerle, A.; Krivec, N.; Benčina, M.; Pisanski, T.; Veličković, T.; Round, A.; Carazo, J. M.; Melero, R.; Jerala, R. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 2017, 35 (11), 1094– 1101,  DOI: 10.1038/nbt.3994
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      Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo
      Ljubetic, Ajasja; Lapenta, Fabio; Gradisar, Helena; Drobnak, Igor; Aupic, Jana; Strmsek, Ziga; Lainscek, Dusko; Hafner-Bratkovic, Iva; Majerle, Andreja; Krivec, Nusa; Bencina, Mojca; Pisanski, Tomaz; Velickovic, Tanja Cirkovic; Round, Adam; Carazo, Jose Maria; Melero, Roberto; Jerala, Roman
      Nature Biotechnology (2017), 35 (11), 1094-1101CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest contg. >700 amino-acid residues and measuring 11 nm in diam. Their stability and folding kinetics were similar to those of natural proteins. Soln. small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophys. anal. confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.
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      Seuring, J.; Agarwal, S. Polymers with upper critical solution temperature in aqueous solution. Macromol. Rapid Commun. 2012, 33 (22), 1898– 920,  DOI: 10.1002/marc.201200433
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      Polymers with Upper Critical Solution Temperature in Aqueous Solution
      Seuring, Jan; Agarwal, Seema
      Macromolecular Rapid Communications (2012), 33 (22), 1898-1920CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. This review focuses on polymers with upper crit. soln. temp. (UCST) in water or electrolyte soln. and provides a detailed survey of the yet few existing examples. A guide for synthetic chemists for the design of novel UCST polymers is presented and possible handles to tune the phase transition temp., sharpness of transition, hysteresis, and effectiveness of phase sepn. are discussed. This review tries to answer the question why polymers with UCST remained largely underrepresented in academic as well as applied research and what requirements have to be fulfilled to make these polymers suitable for the development of smart materials with a pos. thermoresponse.
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      Dignon, G. L.; Zheng, W.; Mittal, J. Simulation methods for liquid-liquid phase separation of disordered proteins. Curr. Opin Chem. Eng. 2019, 23, 92– 98,  DOI: 10.1016/j.coche.2019.03.004
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      Simulation methods for liquid-liquid phase separation of disordered proteins
      Dignon Gregory L; Mittal Jeetain; Zheng Wenwei
      Current opinion in chemical engineering (2019), 23 (), 92-98 ISSN:2211-3398.
      Liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and other biomolecules is a highly complex but robust process used by living systems. Drawing inspiration from biology, phase separating proteins have been successfully utilized for promising applications in fields of materials design and drug delivery. These protein-based materials are advantageous due to the ability to finely tune their stimulus-responsive phase behavior and material properties, and the ability to encode biologically active motifs directly into the sequence. The number of possible protein sequences is virtually endless, which makes sequence-based design a rather daunting task, but also attractive due to the amount of control coming from exploration of this variable space. The use of computational methods in this field of research have come to the aid in several aspects, including interpreting experimental results, identifying important structural features and molecular mechanisms capable of explaining the phase behavior, and ultimately providing predictive frameworks for rational design of protein sequences. Here we provide an overview of computational studies focused on phase separating biomolecules and the tools that are available to researchers interested in this topic.
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      Chou, H. Y.; Aksimentiev, A. Single-Protein Collapse Determines Phase Equilibria of a Biological Condensate. J. Phys. Chem. Lett. 2020, 11 (12), 4923– 4929,  DOI: 10.1021/acs.jpclett.0c01222
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      Single-Protein Collapse Determines Phase Equilibria of a Biological Condensate
      Chou, Han-Yi; Aksimentiev, Aleksei
      Journal of Physical Chemistry Letters (2020), 11 (12), 4923-4929CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Recent advances in microscopy of living cells have established membraneless organelles as crit. elements of diverse biol. processes. The body of exptl. work suggests that formation of such organelles is driven by liq.-liq. phase sepn., a phys. process that has been studied extensively for both simple liqs. and mixts. of polymers. Here, we combine mol. dynamics simulations with polymer theory to show that the thermodn. behavior of one particular biomol. condensate-fused in sarcoma (FUS)-can be quant. accounted for at the level of the chain collapse theory. First, we show that a particle-based mol. dynamics model can reproduce known phase sepn. properties of a FUS condensate, including its crit. concn. and susceptibility to mutations. Next, we obtain a polymer physics representation of a FUS condensate by examg. the behavior of a single FUS protein as a function of temp. We use the chain collapse theory to det. the thermodn. properties of the condensate and to characterize changes in the single-chain conformation at the onset of phase sepn. Altogether, our findings suggest that the phase behavior of FUS condensates can be explained by the properties of individual FUS proteins and that the change in the FUS conformation is the main force driving for the phase sepn.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXpslygtb0%253D&md5=4dcc89c049c013265c47d5fa3357b55e
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      Karginov, A. V.; Zou, Y.; Shirvanyants, D.; Kota, P.; Dokholyan, N. V.; Young, D. D.; Hahn, K. M.; Deiters, A. Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J. Am. Chem. Soc. 2011, 133 (3), 420– 3,  DOI: 10.1021/ja109630v
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      Light Regulation of Protein Dimerization and Kinase Activity in Living Cells using Photocaged Rapamycin and Engineered FKBP
      Karginov, Andrei V.; Zou, Yan; Shirvanyants, David; Kota, Pradeep; Dokholyan, Nikolay V.; Young, Douglas D.; Hahn, Klaus M.; Deiters, Alexander
      Journal of the American Chemical Society (2011), 133 (3), 420-423CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We developed a new system for light-induced protein dimerization in living cells using a photocaged analog of rapamycin together with an engineered rapamycin binding domain. Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFKqtbbI&md5=e0f7a37d60da92696f04b8513d9091b3
    74. 74
      Heidenreich, M.; Georgeson, J. M.; Locatelli, E.; Rovigatti, L.; Nandi, S. K.; Steinberg, A.; Nadav, Y.; Shimoni, E.; Safran, S. A.; Doye, J. P. K.; Levy, E. D. Designer protein assemblies with tunable phase diagrams in living cells. Nat. Chem. Biol. 2020, 16 (9), 939– 945,  DOI: 10.1038/s41589-020-0576-z
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      Designer protein assemblies with tunable phase diagrams in living cells
      Heidenreich, Meta; Georgeson, Joseph M.; Locatelli, Emanuele; Rovigatti, Lorenzo; Nandi, Saroj Kumar; Steinberg, Avital; Nadav, Yotam; Shimoni, Eyal; Safran, Samuel A.; Doye, Jonathan P. K.; Levy, Emmanuel D.
      Nature Chemical Biology (2020), 16 (9), 939-945CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Protein self-organization is a hallmark of biol. systems. Although the physicochem. principles governing protein-protein interactions have long been known, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-sepd. compartments, remain challenging to characterize. To illuminate such principles, the authors create a system of two proteins designed to interact and form mesh-like assemblies. The authors devise a new strategy to map high-resoln. phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their mol. properties, enabling the authors to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equil. effects seen in growing cells. Finally, cotranslational protein binding suffices to recruit a mRNA to the designed micron-scale structures.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlynt7nM&md5=4fe65ded15ab296cbf0ce4ccaf6b073a
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      Roberts, S.; Harmon, T. S.; Schaal, J. L.; Miao, V.; Li, K. J.; Hunt, A.; Wen, Y.; Oas, T. G.; Collier, J. H.; Pappu, R. V.; Chilkoti, A. Injectable tissue integrating networks from recombinant polypeptides with tunable order. Nat. Mater. 2018, 17 (12), 1154– 1163,  DOI: 10.1038/s41563-018-0182-6
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      Injectable tissue integrating networks from recombinant polypeptides with tunable order
      Roberts, Stefan; Harmon, Tyler S.; Schaal, Jeffery; Miao, Vincent; Li, Kan; Hunt, Andrew; Wen, Yi; Oas, Terrence G.; Collier, Joel H.; Pappu, Rohit V.; Chilkoti, Ashutosh
      Nature Materials (2018), 17 (12), 1154-1163CODEN: NMAACR; ISSN:1476-1122. (Nature Research)
      Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helixes, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temps. Through coarse-grain simulations, we show that hysteresis arises from phys. crosslinking due to mesoscale phase sepn. of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temp., they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFajsL3N&md5=8bea1a55180b35733f5a0e1e5f3fba0d
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      Hastings, R. L.; Boeynaems, S. Designer Condensates: A Toolkit for the Biomolecular Architect. J. Mol. Biol. 2021, 433 (12), 166837,  DOI: 10.1016/j.jmb.2021.166837
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      Designer Condensates: A Toolkit for the Biomolecular Architect
      Hastings, Renee L.; Boeynaems, Steven
      Journal of Molecular Biology (2021), 433 (12), 166837CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      A review. Protein phase sepn. has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomol. condensates. While the implication of this phys. phenomenon within cell biol. is providing us with novel ways for understanding how cells compartmentalize biochem. reactions and encode function in such liq.-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biol. matter. Here, we propose that understanding the cell's instruction manual of phase sepn. will enable bioengineers to begin creating novel functionalized biol. materials and unprecedented tools for synthetic biol. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the phys. driving forces underlying phase sepn., with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalog and construction manual for the aspiring (membraneless) biomol. architect. Our approach aims to inspire a new generation of bioengineers to rethink phase sepn. as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biol. function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjs1ygu7s%253D&md5=d047044e898b2072909babd94b2aa8ce
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      Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Reversible hydrogels from self-assembling artificial proteins. Science 1998, 281 (5375), 389– 92,  DOI: 10.1126/science.281.5375.389
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      Reversible hydrogels from self-assembling artificial proteins
      Petka, Wendy A.; Hardin, James L.; McGrath, Kevin P.; Wirtz, Denis; Tirrell, David A.
      Science (Washington, D. C.) (1998), 281 (5375), 389-392CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Recombinant DNA methods were used to create artificial proteins that undergo reversible gelation in response to changes in pH or temp. The proteins consist of terminal leucine zipper domains flanking a central, flexible, water-sol. polyelectrolyte segment. Formation of coiled-coil aggregates of the terminal domains in near-neutral aq. solns. triggers formation of a three-dimensional polymer network, with the polyelectrolyte segment retaining solvent and preventing pptn. of the chain. Dissocn. of the coiled-coil aggregates through elevation of pH or temp. causes dissoln. of the gel and a return to the viscous behavior that is characteristic of polymer solns. The mild conditions under which gel formation can be controlled (near-neutral pH and near-ambient temp.) suggest that these materials have potential in bioengineering applications requiring encapsulation or controlled release of mol. and cellular species.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXkslagu74%253D&md5=c8690002b3db06a74f25f4574f37d231
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      Kelley, F. M.; Favetta, B.; Regy, R. M.; Mittal, J.; Schuster, B. S. Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (51), e2109967118  DOI: 10.1073/pnas.2109967118
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      Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants
      Kelley, Fleurie M.; Favetta, Bruna; Regy, Roshan Mammen; Mittal, Jeetain; Schuster, Benjamin S.
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (51), e2109967118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Cells contain membraneless compartments that assemble due to liq.-liq. phase sepn., including biomol. condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct compn., such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liq. phases of distinct compns. and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biol. function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomol. condensates. We designed and examd. families of amphiphilic proteins comprising one phase-sepg. domain and one non-phase-sepg. domain. In particular, these proteins contain the sol. structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concn. resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concns. coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these exptl. observations, supported by mol. simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomol. condensates.
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  • Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.2c00250.

    • Additional experiments including varying concentrations and pH of mixtures of RGG tagged with coiled coils, FRAP experiments, quantification of yeast data, and legends describing supplemental movies (PDF)

    • Bright-field and 488 nm channel images of condensate formation from 10 μM RGGFKBP and RGG-FRB (AVI)

    • Condensate formation in cell-sized emulsions (AVI)

    • Rapamycin induced condensate formation in live yeast cells (AVI)


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