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Design of Protein Logic Gate System Operating on Lipid Membranes
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  • Neža Omersa
    Neža Omersa
    Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
    Biomedicine Doctoral Program, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
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  • Saša Aden
    Saša Aden
    Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
    Biomedicine Doctoral Program, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
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  • Matic Kisovec
    Matic Kisovec
    Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
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    Orcidhttp://orcid.org/0000-0003-4131-2177
  • Marjetka Podobnik
    Marjetka Podobnik
    Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
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  • Gregor Anderluh*
    Gregor Anderluh
    Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
    *Email: [email protected]
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    Orcidhttp://orcid.org/0000-0002-9916-8465
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ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2020, 9, 2, 316–328
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https://doi.org/10.1021/acssynbio.9b00340
Published January 29, 2020

Copyright © 2020 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein–membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein–membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.

Copyright © 2020 American Chemical Society

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Synthetic biology is a rapidly developing field providing a wide variety of options for construction of biomolecular computers in vitro and in vivo with numerous applications in nanotechnology. (1−3) Diverse systems have been developed employing mostly DNA, (4−8) but also RNA, (9) or enzymes (10−15) to execute logic computing tasks using biomolecule-based Boolean logic gates. While a majority of the work has been done at the DNA or protein level, the engagement of lipid membranes and membrane proteins has been very limited in molecular-scale computational elements (16−19) due to particular structural features of membrane proteins and the complex nature of the lipid bilayer membrane. (20)
Protein logic gates consist of an input component, which is sensitive to specific input signal, and an output component, which upon transduction of the incoming signal produces the perceivable effect. (21) Engineering principles are very diverse as protein logic functions can be achieved with either single domain proteins, where the same domain has the input and the output capability, or by fusing two domains where one domain functions as a recognition domain (the input domain) and the other as an effector (the output domain). (22) Proteins with a natural ability to form defined pores in membranes, so-called pore-forming proteins or pore-forming toxins (PFTs), offer an excellent option for logic gate design on membranes. These protein molecules are soluble as monomeric units, capable of binding to lipid membranes in a lipid-specific manner and consequently form oligomeric transmembrane pores, which are well-defined in terms of shape and size. (23,24) Pore-formation is a complex process and is composed of succession of steps that can be manipulated in order to control the pore opening. (25,26) Several applications of PFTs have been developed, e.g., for release of compounds encapsulated in lipid vesicles-based delivery systems, (27) biosensing, (28−32) and as integral parts of artificial cells. (19,33,34) However, all those systems lack self-regulatory modules or produce only small (around 2 nm in diameter) membrane pores.
To bypass limitations of existing protein-vesicle systems, we present a unique multilevel system where we combined synthetic biology with the rational design and directed evolution based on a PFT listeriolysin O (LLO). This protein toxin is a major virulence factor of bacteria Listeria monocytogenes. It belongs to a protein family of cholesterol-dependent cytolysins that are able to form large pores on membranes of target cells, exceeding 20 nm in diameter. (35) LLO is composed of four domains (36) (Figure 1a), each playing a particular role in the pore-forming process. LLO predominately forms arc-shaped pores on the surface of target cells (37−39) in a succession of steps, which involve binding to lipid bilayers with high cholesterol content by using domain 4 (D4; Figure 1a), oligomerization on the membrane surface, and final pore formation in which two β-hairpins are formed from two clusters of α-helices in domain 3 (D3) of each monomer (Figure 1b). We have recently reported an interesting mutant of LLO, Y406A (Figure 1a), which shows a unique pH-regulated pore-forming activity. While it binds to cholesterol-rich membranes in a wide pH range, it is able to form pores and is thus fully active only at low pH values. (40)

Figure 1

Figure 1. Properties and permeabilizing activity of Y406A. (a) A ribbon model of LLO 3D structure (PDB ID: 4CDB) with each domain (D1–D4) labeled in different color. Position of residue 406 is denoted by an orange color and an arrow. (b) A model of pore formation by Y406A (monomer binding to the membrane, oligomerization, pore formation). Domains are colored as on panel a. (c) Calcein release from large unilamellar vesicles after 30 min is shown at different pH values for LLO (black) and Y406A (red). Data reproduced with permission from Kisovec et al. (40) (d) Relative rate of hemolysis by 2.3 nM CDCs at pH 5.7 in the presence of DARPins: 5 μM D22 (red bars), 5 μM D22M (blue bar), a variant that was used for immobilization to the lipid membrane (see below), and 5 μM D6 and D30 (green bars), DARPin clones that were selected with ribosome display as high affinity binders, but did not inhibit hemolytic activity of Y406A. Mean ± SD; n = 2–7.

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We created a logic gate on lipid membrane by combining Y406A with an additional inhibitor of its activity, a designed ankyrin repeat protein (DARPin) variant 22, D22, which binds reversibly to Y406A. In the off state of the logic gate, Y406A is doubly inhibited by D22 covalently bound to the membrane, and pH > 7.4. The logical functioning of the protein gate was achieved with various cleavages of D22 from the membrane and pH activation of Y406A. Upon system activation, the inhibiting D22 dissociates from Y406A, which then, at a favorable pH, undergoes conformational changes and forms pores in membranes.

Results

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DARPin D22 Binds Specifically to Y406A in Solution and in the Membrane-Bound State

Y406A is an interesting pH-dependent LLO mutant, which has a very narrow activity profile of pH dependence. It is active at low pH values, drastically loses activity in the pH range 6.0–7.4, and is not active at pH values >7.4 (Figure 1c). However, it is still capable of binding to the membrane at high pH values. (40) Y406A is thus perfectly suited for controlled release in liposomal applications employing pH as an input signal. In order to provide another level of control over Y406A permeabilizing activity, we have developed a DARPin-based inhibitor. DARPins represent a useful tool for specific targeting of bigger molecules, such as proteins, due to their large interaction surface and high binding capacity. Specific DARPin inhibitor of permeabilizing activity of Y406A was gained with ribosome display. (41,42) Forty clones among enriched variants after six rounds of ribosome display were isolated and tested with ELISA. Three clones, D6, D22, and D30 that exhibited highest affinity toward immobilized target protein were further selected and checked for permeabilizing activity. D22 was selected for further studies, because of its specific inhibition of hemolytic activity of Y406A, but not of the wild-type LLO (Figure 1d). To further prove the specificity of D22 for Y406A, inhibitory effect of D22 was tested toward another member of cholesterol-dependent cytolysin and a homologue of LLO, Perfringolysin O (PFO) from bacterium Clostridium perfringens, and its mutant Y381A, which is analogous to Y406A (40) (Figure 1d). Indeed, D22 is selective only for Y406A by exhibiting no significant effect on hemolytic activity of these two homologous proteins (Figure 1d).
The size exclusion chromatography proved stable complex formation between Y406A and D22 (Figure 2a). Isothermal titration calorimetry suggested a stoichiometry ratio of Y406A-D22 complex to be 1 (0.96 ± 0.15) (Figure 2b), with a binding constant KD of 114 ± 43 nM (n > 4; average ± SD). LLO did not show any detectable binding at the same conditions (Figure 2b). Small-angle X-ray scattering (SAXS) measurements confirmed formation of Y406A-D22 complex (Figure 2c), indicating that D22 is most likely associated with the domain 2 (D2) of Y406A (Figure 2c inset).

Figure 2

Figure 2. Binding of D22 to Y406A in solution. (a) Size exclusion chromatogram of LLO and Y406A in the absence or presence of D22. Triangles indicate positions of elution peaks for different proteins. Note that LLO travels aberrantly on the size exclusion column eluting with larger volumes of elution buffer than expected. (b) Binding of D22 to LLO (gray) or Y406A (black) in solution (22 mM MES, 150 mM NaCl and 5 mM 2-mercaptoethanol, pH 5.7), measured by isothermal titration calorimetry. Top panel represents raw data of injections of 54.9 μM D22 into a 5.9 μM solution of LLO or Y406A. Bottom panel shows normalized integrated enthalpies plotted against the molar ratio. Circles represent experimental points, and the solid line corresponds to the best fit obtained by one-site reaction model. (c) An overlay of experimental scattering data obtained by SAXS experiment of Y406A-D22 complex (red circles) with the calculated scattering curve from the representative DAMMIF model (χ2 = 1.125, black line). Inset, overlay of Y406A (blue ribbon) and D22 (red ribbon) refined by rigid body modeling (χ2 = 1.12), with the best SAXS bead model in surface representation and the average SAXS bead model in mesh representation.

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In the next step we wanted to test whether D22 interferes with the binding of Y406A to the lipid membrane. LLO and Y406A require high concentrations of lipid receptor cholesterol in membranes, (40,43) and we used a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol 3:2 (mol:mol) in all model lipid vesicles systems presented in this paper, unless stated otherwise. Preincubated mixture of Y406A and D22 in solution showed larger surface plasmon resonance (SPR) response than Y406A alone, which indicates that Y406A-D22 complex can indeed bind to the membrane (Figure 3a). This is in agreement with proposed mode of binding of D22 to Y406A via D2 (Figure 2c), which leaves D4 free for interaction with the lipid membrane. D22 itself did not bind to the lipid membrane (Figure 3a). D22 also bound to the membrane-bound Y406A and the interaction was reversible, with slow dissociation of D22. As expected, D22 did not bind to the membrane-bound LLO (Figure 3b). This was also independently confirmed by vesicle sedimentation assays (Figures 3c and d). Thus, D22 specifically binds to soluble as well as membrane-bound Y406A.

Figure 3

Figure 3. Interaction of D22 with Y406A in the lipid membrane environment. (a) SPR measurements showing binding of 100 nM Y406A, 5 μM D22 and preincubated Y406A-D22 complex (with same concentrations of individual proteins as used for single proteins injections) to large unilamellar vesicles. (b) SPR sensorgrams of 5 μM D22 binding to 100 nM membrane-inserted Y406A or LLO. Membrane denotes control experiment with D22 binding to vesicles only. (c) Vesicle sedimentation assays with multilamellar vesicles after preincubation of LLO and D22, or Y406A and D22 in solution (“preincubated”) or when LLO or Y406A were first preincubated with vesicles (“membrane bound”). p, pellet; s, supernatant; w, additional washing step, which was included when assaying membrane bound LLO or Y406A in order to check for the completeness of binding. Band at app. 60 kDa corresponds to LLO or Y406A, while band at ca. 18 kDa corresponds to D22. D22 is present at 5× molar excess; therefore, a large portion of it is always unbound in supernatant. (d) Quantification of the SDS-PAGE data from (c) by densitometry. Full binding of D22 to Y406A (100%) was considered when one-fifth of the applied D22 was bound to Y406A. Mean ± SD; two sample t test, ***P < 0.001 (n = 4–6). Amount of bound D22 is reported when preincubated with LLO (1) or Y406A (2) in solution or when LLO (3) or Y406A (4) were first bound to vesicles.

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D22 Provides Additional Control of Y406A Permeabilizing Activity

We next verified ability of D22 to provide additional control of Y406A permeabilizing activity in different functional assays. For this purpose, we assayed permeability of giant unilamellar vesicles (GUVs) for fluorescently labeled dextran of 10 kDa size (FD10) and a Stokes radius of 23.6 Å at two pH values, 6.5 and 8.0. LLO was able to permeabilize GUVs at either pH and was not affected by D22 (Figure 4a). Y406A clearly showed activity only at low pH and in the absence of D22, while in the presence of D22, the permeabilizing activity of Y406A was inhibited (Figure 4b). We also assayed hemolytic activity of LLO or Y406A at two pH values, pH 5.7 and 7.4, and in the presence or absence of D22. Hemolysis results confirmed the results obtained in the GUVs system (Figures 4c and d). D22 itself was not able to induce any damage to the GUV lipid membrane at either pH (Figure 3e) and was not hemolytic up to 9.5 μM concentration (data not shown), which is in agreement with SPR results that showed a lack of membrane association for D22 (Figure 3). The experiments presented in Figure 4 convincingly show that activity of Y406A can be controlled by pH as expected, (40) as well as with D22, which provides an additional level of control over Y406A activity. This system can thus conceptually be described as the NOR logic gate, when activity (pore formation) is observed only in the absence of the two signals, D22 and pH > 7.4 (Figure 4f).

Figure 4

Figure 4. Modulation of Y406A permeabilization activity by D22 and pH. (a) Permeabilization of GUVs for FD10 at different conditions and induced by the 50 nM LLO. (b) Permeabilization induced by 50 nM Y406A. The graphs on the right in A and B show quantification of GUVs data from confocal microscopy images as represented on the left. Mean ± SD; n = 96–568. (c) Hemolysis induced by 18.2 nM LLO at different conditions. (d) Hemolysis induced by 18.2 nM Y406A. In C and D, mean ± SD is presented; n = 3. (e) 5 μM D22 by itself does not induce permeabilization of GUVs. Confocal images of GUVs on the left, and quantification is presented on the right. n is 256 and 458 for pH 6.5 and 8.0, respectively. (f) The truth table and schematic representation of a NOR logic gate for the Y406A-D22 system.

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We further demonstrated the principle of selective inhibition of Y406A permeabilizing activity by assaying release of encapsulated fluorescent probe calcein from small and large unilamellar vesicles (SUVs and LUVs, respectively). In a SUVs system we essentially obtained similar results as in the GUVs system, with LLO showing similar activity regardless the condition at which it was assayed (Figure 5a), while Y406A only showed activity at pH 6.5 in the absence of D22 (Figure 5b). Y406A showed negligible release of calcein also in a LUVs system when assayed at high pH (8.0) (Figure 5c, traces A and D). Membrane-bound Y406A can be effectively activated by addition of small quantity of HCl (Figure 5c, blue trace D, pH drop to approximately 6.5) in agreement with previous work. (40) D22 provided an effective control over Y406A permeabilizing activity also in this system. The pH activation of Y406A did not occur when D22 was added to solution containing vesicles and Y406A (Figure 5c, trace A), when Y406A was preincubated with D22 before addition to vesicles (Figure 5c, trace B) or when Y406A was added to vesicles and D22 in an acidic environment subsequently (Figure 5c, trace C). Interestingly, addition of D22 after activation of Y406A also inhibited further release of calcein, showing that D22 is efficient modulator that rapidly affects permeabilizing activity of Y406A (Figure 5c, trace E). In summary, permeabilizing experiments employing four independent model systems thus clearly exemplify a two-level control system of Y406A activity, modulated by the change in pH and the presence of D22.

Figure 5

Figure 5. Modulation of Y406A activity in calcein release experiments. (a,b) Calcein release from SUVs composed of POPC:Chol, 3:2 (mol:mol) as a result of pore formation by 1 μM LLO (a) or Y406A (b), in presence or absence of 5 μM D22 and at pH values 6.5 and 8.0. (c) Calcein release from LUVs composed of POPC:Chol, 1:1 (mol:mol) monitored at different conditions. Vesicles were stirred in 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 8.0. Final concentration of 30 nM Y406A (black triangle), 1 μM D22 or 3 μL of 7% HCl (to reduce pH to approximately 6.5) were added, respectively, at times denoted by triangles. The scale bar is the same for all fluorescent traces.

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Membrane-Based System Including Covalently Bound D22 Allows Employment of Various Signals for Activation of Y406A

We showed that D22 does not associate with lipid membrane by itself (Figure 3) and does not damage the lipid membrane (Figures 4 and 5), which is a convenient feature for development of logic gates, since it could be attached to the lipid bilayer without damaging or changing its properties. In order to bind D22 to lipid membranes, we prepared a variant of D22 by adding the matrix metalloproteinase 9 (MMP-9) cleavable peptide and an additional cysteine to the C-terminal end (D22M). This change in amino acid sequence did not affect inhibitory potential of D22M toward Y406A (Figure 1d). Such construct allowed attachment of D22 to the lipid membrane by conjugation via the introduced cysteine residue at the C-terminus and offered removal of D22 from the membrane surface by either proteolytic cleavage or addition of a reductant. Two alternative conjugations of D22M to lipid bilayers were thus prepared by using different functionalized lipids. In the case of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000Mal), D22 was attached to the lipid via stable irreversible thioether linkage and the subsequent release of D22 was achieved using MMP-9 cleavage (Figure 6a). In the case of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] (DSPE-PEG2000PDP), D22M was attached to the lipid via reversible S–S bond and the subsequent release of D22 was achieved upon addition of a reducing agent such as Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Figure 6c). Both lipids contain a PEG linker, which provides flexibility of membrane-anchored D22M needed for efficient inhibition of membrane-bound Y406A.

Figure 6

Figure 6. Conjugation of D22M to vesicles and cleavage with different agents. Schematic diagrams of used systems and structures of employed lipids are shown. Y406A is presented with green color, D22 is shown in orange. Different parts of lipids used for conjugation are presented with different colors in the structural formulas and on the diagram. (a–c) A system employing DSPE-PEG2000Mal lipid, which attaches D22 to the lipid membrane and allows subsequent cleavage by MMP-9. (d–f) A system employing DSPE-PEG2000PDP lipid, which allows cleavage with reductant TCEP. The input of different proteins and reagents is shown above the sedimentation assay gels. The approximate positions of different proteins and lipids on the gels are indicated by arrows. i, 2 μg of D22M as an input; p and s denote pellet and supernatant after centrifugation of MLVs, respectively. (c) and (f) each represent one experiment where MLVs were first incubated with Y406A and then with MMP-9 or TCEP. 1, pellet after both incubations; 2, supernatant after centrifugation of MLVs after incubation with Y406A; 3, supernatant after centrifugation of MLVs with bound Y406A and incubation of MMP-9 or TCEP.

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We optimized assays by (i) selecting the appropriate variant of D22 (N- terminal or C-terminal modification) and functionalized lipids (we tested lipids with or without flexible PEG linker) and (ii) determining the minimal proportion of functionalized lipids to show efficient inhibition of Y406A and cleavage of conjugated D22M from the MLVs by either MMP-9 or TCEP. We tested a range of Y406A (200 nM to 2 μM), conjugated lipids (from 2 to 10 mol %), TCEP (10 to 30 mM) and MMP-9 (0.002 μg, 0.02 μg and 0.1 μg) concentrations. We observed that the most optimal concentrations were the following, 500 nM Y406A, 2 mol % of conjugated lipids, 10 mM TCEP or 0.1 μg of MMP-9. When we used higher concentration of conjugated lipid, we needed more Y406A for efficient permeabilization, but also more TCEP or MMP-9, which we tried to avoid because of background effects of buffers in which these substances are stored and unwanted cleavage of proteins by MMP-9 (see below). After D22M conjugation, stability of D22M attachment to MLVs and cleavage from vesicles by different agents was checked by SDS-PAGE analysis (Figure 6). D22M was in both cases attached to the surface of vesicles and was successfully released from them after addition of either MMP-9 (Figure 6b) or TCEP (Figure 6e). We also checked whether membrane-conjugated D22M could be released from the membrane upon incubation of D22M-conjugated vesicles with Y406A. Indeed, the majority of the D22M was released from the vesicle membrane upon MMP-9 or TCEP cleavage with only minor amounts of D22M visible on the SDS-PAGE gels in the pellet fraction (Figures 6c and f), which might be due to the fact that not all of the D22M dissociated from Y406A despite the fact it was cleaved from the vesicles. On the other hand Y406A was not affected by such treatments and remained stably associated with the lipid membrane (Figures 6c and f), apart from minor degradation of Y406A in the pelleted fraction in the presence of MMP-9 (Figure 6c), which, however, did not have a great effect on Y406A’s ability to form pores (see below).

Membrane System with Logic Gate Based on Y406A and D22M

Conjugation of D22M to the lipid membrane of vesicles allowed development of a controllable system with different logic gates (Figure 7a). We followed calcein release from the vesicles to prove the gate opening (i.e., pore formation) of different systems. To develop an AND gate, D22M was attached by maleimide conjugation using DSPE-PEG2000Mal, and calcein was released from MLVs only upon both low pH and MMP-9 cleavage (Figure 7b). In the second system, an OR-AND gate could be conceptualized (Figure 7c). Employment of DSPE-PEG2000PDP was used to attach D22M to the vesicles, and the release of D22M from the vesicles was achieved by either the reducing agent or MMP-9, whereas system activation was achieved when one or both of those signals were present in addition to low pH in the surrounding buffer solution. This experiment was performed in microplate wells with ordered addition of different reagents and no possibility for washing away the components present in the reaction mixture. However, robust and significant differences between the closed (on) and the opened (off) states were found in both cases (Figure 7).

Figure 7

Figure 7. A tunable vesicle system. (a) Y406A activity is controlled by pH and the reversible inhibitor D22. Vesicles are at pH 8.0; therefore, two input signals are needed for system activation: lowering pH with HCl and elimination of D22 from the system by release from vesicles induced by MMP-9 or TCEP. (b) A system based on DSPE-PEG2000Mal lipid. Bars represent fluorescence increase due to release of calcein from MLVs. A logic truth table and schematic representation of an AND gate is presented below the graph. (c) A system based on DSPE-PEG2000PDP lipid, which gives more flexibility for the removal of D22 from the vesicle membrane. A logic truth table and schematic representation of an OR-AND gate is presented beside the graph. n = 3–6, mean ± SD *P < 0.05; **P < 0.01.

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Discussion

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Existing protein logic gates are generally operated via proteolytic cleavage (11,15) or allosteric regulation of conformationally stable proteins. Complex protein structures, enabling multidomain gating, are implemented in order to obtain higher efficiency of the system. (10,11,13,21,44−47) However, a majority of the work on protein logic gates is done with enzymes in solution. A valuable contribution to synthetic biology represent PFTs, (24) which are an excellent system since they are well characterized at the structural level, form stable pore complexes that are hard to dissociate once assembled, operate at the level of lipid membranes, and are amenable for rational design and mutagenesis to yield variants with changed useful properties. The incorporation of a stimuli-sensing PFT into lipid vesicles could thus enable a controlled opening of the membrane and consequently release of the encapsulated content at the desired site.
In this work we used a mutant of the PFT LLO, Y406A, to design protein logic gate at the surface of lipid membranes. Y406A is perfectly suited for such applications, since it forms transmembrane homo-oligomeric pores from soluble monomeric units in a controllable stepwise mechanism. We have shown before (40) that single amino acid mutation in LLO D2, Y406A, drastically alters protein characteristics, most significantly its pH-dependent behavior, whereas the size of the formed pores remains generally the same. Here we employed a small protein molecule D22, which specifically and reversibly inhibits activity of Y406A by binding to the domain D2. The complex Y406A-D22 can still bind to lipid membranes (Figure 3); however, Y406A in such complex is not able to form pores. Our results suggest that D22 binds to the D2 of Y406A, which might prevent conformational changes in Y406A needed in the final stages of pore formation. (48) An additional advantage of LLO-based systems is that LLO forms large pores with diameter of 25–40 nm in lipid membranes. This feature makes it extraordinary for synthetic biology and could be used for example in liposome-based delivery systems of larger proteins. In comparison to small pores of approximately 1.5–2 nm in diameter that are formed by some toxins, such as α-toxin from Staphylococcus aureus (49) and aerolysin-like proteins such as lysenin, (50) Y406A can make much larger pores enabling release of larger compounds, such as 10 kDa dextran shown in Figure 4, or even larger 70 kDa dextran (∼12 nm in diameter). (40) Y406A-D22 system could thus be used for liposome-based delivery of small proteins, such as nanobodies, or small enzymes.
We developed protein logic functioning for precise activation and release of lipid vesicles-encapsulated cargo. Our robust system consists of a vesicle with membrane-conjugated D22 that binds and reversibly inhibits Y406A. The system can be activated upon two different and very specific signals (low pH in conjunction with reductive environment or MMP-9), and therefore opens the vesicle in regulated manner and release the vesicle-encapsulated cargo. The nature of the system follows the “all-or-nothing” principle, where the response is fully expressed once activated. Two-level complex gates, especially with the possibility of different activating inputs that do not cancel each other out, function much better due to higher efficiency and simplicity compared to the sum of the individual gates. The modularity makes the logical functioning of this system suitable for diverse applications because of distinct operations being executed by separate components and the ease of expansion of input signals by incorporation of recognition sites for other proteolytic enzymes at the C-terminus of D22, or integration of other cleavable linkers, responsive to various reagents (reducing, oxidizing, nucleophilic/basic, electrophilic/acidic), metals, lysosomal enzymes, light, etc. (51,52) Further development of logic gate system is also possible with utilization of other LLO variants. LLO undergoes extensive conformational rearrangements during pore formation, for example two clusters of helices in D3 rearrange and form two β-hairpins that are inserted in lipid membrane and form the β-barrel of the final pore (Figure 1b). Introduction of two cysteine residues in one of the helix clusters, A318C-L334C, prevents these rearrangements and pore formation upon disulfide formation. (53) Such LLO variants could help to achieve additional logic operations using reductants as one of the input signals.
The presented system has a high potential to be used for therapeutic purposes, as it enables vesicle packing for directed administration of small molecules, biologics, or other protein or nonprotein cargos of high molecular weight, enabling very precise regulation and reliable delivery. The provided system has a potential in cancer treatment, where especially the differences in pH, (54) redox potential, (55) and presence of metalloproteinases (56) between normal and cancerous tissue are significant (57) and could provide input signals for the Y406A-D22 system.

Methods

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Materials

The plasmid pRDV was kindly provided by dr. Plückthun’s lab. (42,58) DARPin gene library was obtained from Eurofins Genomics, Germany. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000Mal) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyl)-dithiopropionyl (polyethylene glycol)-2000] (DSPE-PEG2000PDP) were from Avanti Polar Lipids (Alabaster, United States). Lissamine Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine DHPE) was purchased from Invitrogen (United States). Matrix Metalloproteinase 9 (MMP-9) was from Merck, Germany. All other chemicals were from Sigma, United States, unless stated otherwise.

D22 Design and Ribosome Display

The inhibitor that potently, specifically and selectively binds to Y406A, D22, was developed via directed evolution approach by using a randomized gene library of human ankyrin repeat consensus sequence. (59) It contains 2 capping and 3 modular ankyrin repeats, each repeat forming a β-sheet followed by two α-helices. We applied diversity to the elements of secondary structure by randomizing 20 amino acid residues per each of 3 internal modular repeats: on the β-sheet and on the first α-helix. The resulting DARPin naïve library was cloned into pRDV vector and subjected to 6 rounds of ribosome display selection technique, following the protocol of Dreier and Plückthun, (41) with LLO directly immobilized on plastic surface as a target (concentration decreasing from 200 nM at first round to 20 nM to sixth round of selection). Selection pressure was performed with the following washing steps: 5 short washes at the first round, gradually enhancing to 5 × short, 2 × 20 min, 40 min, 1 short wash at the sixth round.

Cloning, Expression, and Purification of LLO, Y406A, D22, and D22M

Genes for the wild-type LLO, Y406A, or D22 were multiplied by PCR, cleaved and inserted into a precleaved pProEXHTb expression vector. DARPin 22M (D22M) was created by substituting Cys50 and Cys167 to Ser (to avoid unspecific conjugation) and extending the protein at the C-terminal end with MMP-9 cleavage site sequence GPLGMLSQ, (60) followed by the GGGSGGGS linker and the final residue Cys (for specific conjugation to the lipids). The gene for D22M was inserted into the precleaved pET28a expression vector. All constructs were verified by nucleotide sequencing. Expression of genes was performed in Escherichia coli BL21 (DE3) pLysS strain (Novagen, United States) in terrific broth (TB) supplemented with ampicillin. Cells were grown at 37 °C with shaking until optical density at 600 nm reached ∼1. The expression of desired genes was then induced with 0.5 mM IPTG (final concentration). Cells were grown for additional 5 h at 37 °C (D22) or 20 h at 20 °C, respectively (LLO and Y406A), centrifuged for 10 min at 2800g at 4 °C and frozen to −20 °C. Buffer containing 50 mM Na-phosphate, 250 mM NaCl, 5 mM 2-Mercaptoethanol and 2 mM phenylmethylsulfonyl fluoride (PMSF) at pH 6.5 was added to thawed lysate, which was then sonicated and centrifuged for 1 h at 35 000g at 4 °C. Supernatants were filtered through 0.45 and 0.22 μm filters. Filtrates were purified by immobilized metal affinity chromatography (IMAC), using a 9.6-ml Ni-NTA column (Qiagen, Germany), coupled to the Åkta FPLC system (Amersham Biosciences, United Kingdom). Column was equilibrated with buffer, containing 50 mM Tris-HCl, 500 mM NaCl at pH 7.4 following the application of lysate and washing of unbound proteins with buffer containing 10 mM and 60 mM of imidazole. The bound proteins were eluted by imidazole in the buffer (500 mM for D22 and D22M, and 300 mM for LLO wt and Y406A). His-tags were than cleaved off (0.6 mg Tobacco Etch Virus (TEV) protease to 1 mL of the sample) during overnight dialysis at 4 °C against 50 mM Tris-HCl, 500 mM NaCl at pH 7.4. Flow-through after additional IMAC, containing purified protein, was concentrated and buffer changed to 20 mM MES, 150 mM NaCl, pH 5.7 using Amicon Ultra 10 kDa MWCO (Merck, Germany). Content, size and purity of samples were observed by SDS-PAGE. Fractions containing desired proteins were pooled, concentration determined spectrophotometrically (Agilent 8453 UV–visible Spectroscopy System, United States), aliquoted and stored at −80 °C.

Enzyme-Linked Immunosorbent Assay (ELISA)

100 nM LLO was immobilized on Nunc Maxi-Sorp 96-well microtiter plate (Thermo Fisher, United States) overnight at 4 °C. Wells were washed after that three times with TBST buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween-20 (Merck Millipore, United States), pH 7.4). After that, wells were blocked with buffer containing 0.5% BSA for 1 h and washed three times with TBST. Sample proteins (40 different clones of 5 μM DARPins with hexahistidine tags, isolated after six rounds of ribosome display) were added to wells and incubated for 1.5 h at room temperature with gentle shaking. After that, wells were washed 3 times with TBST and subsequently 1.6 nM mouse monoclonal anti-6His IgG antibodies (Santa Cruz Biotechnology, United States) were added and incubated in wells for 1.5 h at room temperature with gentle shaking. Wells were washed four times with TBST and 10 000 × diluted horseradish peroxidase-conjugated antimouse IgG antibodies (Sigma-Aldrich, United States) were added and incubated for 1 h at room temperature with gentle shaking. Wells were washed four times with TBST. 150 μL of the substrate system for ELISA detection TMB (3,3′,5,5′-tetramethylbenzidine)/H2O2 (Sigma-Aldrich, United States) was added per each well and incubated for 30 min in dark at room temperature. To stop the reaction, 75 μL of TMB substrate for ELISA (Stop solution, Sigma-Aldrich, United States) was added and absorbance was measured immediately with microplate reader Synergy MX (Biotek, United States) at 450 nm and at 600 nm for background subtraction.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography was performed on UPLC Acquity Waters system (Waters Corporation, United States) with Acquity UPLC BEH200 1.7 μm column. The running buffer composition was 10 mM Tris-HCl, 150 mM NaCl, pH 7.4. Samples were injected at 0.4 mL/min flow rate at the concentration of single proteins being 26.6 μM; complexes were mixed at 1:1 molar ratio and preincubated for 30 min at room temperature before the injection. The protein elution profile was monitored with a UV-detector operated at 280 nm.

Isothermal Titration Calorimetry (ITC)

ITC experiments were conducted using a VP-ITC (MicroCal, United States). All protein samples for ITC measurements contained 5 mM 2-mercaptoethanol. 38 injections of 8 μL (first 2 injections 2 μL) of D22 were added by using a computer-controlled microsyringe at intervals of 300 s into the solution of LLO, Y406A, or buffer under constant stirring (307 rpm) at 25 °C. Reference power was set to 10 μcal/s and initial delay before injections was 180 s. The concentrations used for the experiments were 5.9 μM for LLO and Y406A, and 54.9 μM for D22 (the concentration in the syringe). Titrations were carried out in the buffer containing 22 mM MES, 150 mM NaCl and 5 mM 2-mercaptoethanol, at pH 5.7. Buffer was the same for all components to cancel out the possibility of the buffer background signal. Binding parameters were calculated by integration of individual titration peaks and presenting the resulting binding isotherm in a Wiseman plot. Isotherm was fitted to a model for one set of sites, which works for one site or n identical sites, binding constant (K) and enthalpy (ΔH). We calculated stoichiometry (n), ΔH and K for each data set using the one-site reaction model in the Origin 7 ITC software package (MicroCal, USA).

Small-Angle X-ray Scattering (SAXS) Data Collection, Ab Initio Shape Determination and Molecular Modeling

Y406A and D22 were mixed in a 1:1 molar ratio and stored at −70 °C. Buffer composition was 20 mM MES, 150 mM NaCl, 1 mM DTT, pH 5.7. After thawing, the sample was centrifuged and serial dilution from 164 μM to 5.5 μM was prepared. SAXS measurements were performed at the high brilliance synchrotron beamline P12 at the European Molecular Biology Laboratory (EMBL, DESY, Hamburg). The X-ray wavelength was λ = 1.24 Å. Detector (Pilatus 2M) to sample distance was 3 m, resulting in a q range of 0.003–0.5 Å–1. Measurements were performed at 23 °C, samples storage temperature before the measurement was 10 °C. Exposure time was 45 ms and 20 frames were recorded in one second with constant flow through the quartz capillary. The flow ensures that a certain sample volume is illuminated only for a certain time and is displaced by the fresh sample volume. On the basis of the comparison of successive frames, no detectable radiation damage was observed. Frames were normalized to the transmitted beam intensity, azimuthally integrated and averaged, resulting in scattering curves l(q) versusq. The background from the quartz capillary and sample buffer was subtracted. These averaged and subtracted difference curves were next normalized by their concentrations. Low quality data and very low and very high q values were discarded. Valid q range was between 0.006 and 0.48 Å–1. Two approaches to data analysis were tested. First, concentrations from 37.5 μM to 5.5 μM were used to extrapolate data and in parallel concentrations of 73 μM (high-s) and 9.7 μM (low-s) were merged for modeling. We saw no major differences between extrapolated data and merged data and we report data from the merged data set. All data manipulations were performed with PRIMUS, part of ATSAS software suite. (61) Low-resolution shape envelopes were determined using the ab initio bead modeling program DAMMIF (62) as part of the ATSAS online software suite. The results of 20 independent DAMMIF runs were clustered and analyzed online using DAMAVER (63) to identify the most representative models. Rigid body molecular modeling was conducted using in silico mutated crystal structures of Y406A and D22. LLO (PDB ID: 4CDB (36)) residue Tyr406 was mutated to Ala using Pymol. (64) First 19 residues of LLO were absent in the crystal structure and were modeled using Modeler software (65) to neutralize the difference between the measured and modeled Y406A. D22 was modeled with SWISS-MODEL (66) and model PDB ID: 2P2C (67) was used as the template. Rigid body modeling was conducted in ATSAS online service using the program SASREF. (68) Merged data set and two subunits were inserted and modeled without any symmetry. Models were aligned in Pymol with SASpy plugin (supalm). (69)

Lipid Vesicles Preparation

POPC and cholesterol were dissolved in chloroform and mixed together in a molar ratio 3:2 or 1:1. The solvent was removed under reduced pressure by rotary evaporator (Büchi, Switzerland) and the resulting lipid film on glass flask wall was left to dry for additional 4 h in a vacuum (SpeedVac, Thermo Scientific, United States). The film was then resuspended in the buffer (20 mM MES, 150 mM NaCl, pH 5.7, or 20 mM Tris, 150 mM NaCl, pH 7.4) by alternatingly vortexing and heating (65 °C, water bath) the sample for 10–15 min. To get multilamellar vesicles (MLVs), this sample was additionally freeze–thawed in liquid N2 for 6 times. To obtain SUVs, MLVs were pulse-sonicated on ice using a Cole Parmer CPX500 500 W sonicator for 15 min (10 s on/10 s off duty cycle) at 38% amplitude. MLVs were extruded through 100 nm pore size polycarbonate membranes by the mini extruder (Avanti Polar Lipids, United States) to yield large unilamellar vesicles (LUVs) of 100 nm in diameter. Giant unilamellar vesicles (GUVs) were prepared by electroformation method, as described previously by Ruan et al. (37) Shortly, lipid mixture POPC:Chol 3:2 (mol:mol) with addition of 1 mol % rhodamine-DHPE was placed on ITO-slides and dried under the nitrogen stream. Electroformation was carried out between two conductive ITO-slides (Vesicle Prep Pro, Nanion Technologies, Germany) in sucrose solution (300 mM sucrose, 1 mM MES, pH 6.5, or 300 mM sucrose, 1 mM Tris, pH 8.0) for 4 h with 3 V amplitude and 5 Hz frequency. Afterward, GUVs were sedimented with glucose solution (300 mM glucose, 1 mM MES, pH 6.5, or 300 mM glucose, 1 mM Tris, pH 8.0) and addition of buffer (10 mM MES, 150 mM NaCl, pH 6.5, or 20 mM Tris, 150 mM NaCl, pH 8.0). The osmolarity of all solutions was adjusted with Osmomat 3000 (Gonotec GmbH, Germany).

Surface Plasmon Resonance (SPR)

SPR measurements were performed on a Biacore X100 (GE Healthcare, Biacore AB, Sweden) at room temperature on an L1 sensor chip. Large unilamellar vesicles (LUVs) were loaded on equilibrated sensor chip (20 mM MES, 150 mM NaCl, pH 5.7). Reference flow cell contained LUVs composed only of POPC (representing negative control as LLO requires cholesterol for efficient membrane binding), whereas active flow cell contained LUVs with POPC and cholesterol at 3:2 (mol:mol) ratio. The LUV-coated chip surface was prepared as described. (70,71) All protein samples for SPR measurements contained 10 mM DTT. For studies regarding binding of proteins or protein complexes to membranes, 100 nM Y406A, 5 μM D22, or premixed samples of 5 μM D22 and 100 nM Y406A (incubated for 45 min at room temperature) were injected over the LUV-coated surfaces for 3 min at a flow rate 5 μL/min and dissociated for 20 min. For studies of interactions of D22 with membrane-bound proteins, 100 nM LLO or Y406A was injected over LUV-coated surfaces for 3 min at flow rate 5 μL/min, followed by dissociation for 5 min. Following one blank injection, 5 μM D22 was injected over LLO bound to vesicles for 3 min at a flow rate 5 μL/min and left to dissociate for 20 min. Blank injections were subtracted from sample sensorgrams to eliminate the influence of buffer and DTT. Data was processed with Biaevaluation v3.2 (GE Healthcare, United Kingdom) software. Three individual experiments were conducted for each case.

Vesicle Sedimentation Assay

Sedimentation assays were carried out in the buffered system (20 mM MES, 150 mM NaCl) at pH 5.7. For studying binding of D22 complexed with binding partner to membranes, protein pairs (LLO + D22 or Y406A + D22) with D22 in 5 × molar excess were first incubated for 15 min at room temperature and then multilamellar vesicles (MLVs) in 2000 × molar excess of lipids were added. For studying interaction of D22 with membrane-inserted LLO or Y406A, LLO or Y406A was first incubated with MLVs for 15 min, followed by addition of D22. Those mixtures were left to incubate for an additional 15 min at room temperature and then centrifuged at 16 100g for 15 min. Supernatants were transferred to fresh microtubes and centrifuged again, whereas pellets were washed with 50 μL of the buffer (resuspended and centrifuged again) and finally resuspended in 15 μL of the buffer. Samples were analyzed in the presence of 10 mM DTT with SDS-PAGE, showing MLV-bound portion in pellet and unbound molecules in supernatant. Proteins were visualized with SimplyBlue SafeStain (Thermo Fisher Scientific, United States) and the amount of bound D22 was determined by densitometry by using ImageJ. (72)

Hemolytic Assay

Bovine erythrocytes, stored at 4 °C in Alsevier preservative, were washed four times with resuspension in the erythrocyte buffer (20 mM MES, 140 mM NaCl, pH 5.7) and centrifugation at 800g for 5 min at room temperature. Erythrocytes were diluted with erythrocyte buffer to yield absorbance of 1.0 at 630 nm, which was determined by microplate reader Synergy MX (Biotek, United States). 50 μL of LLO or Y406A (final concentration 2.3 nM) was mixed with 50 μL of D22 (final concentration 5 μM) and incubated for 45 min at room temperature. Afterward, 100 μL of erythrocyte suspension was added to each well. The final volume in all wells was 200 μL. Absorbance at 630 nm was measured every 20 s for 20 min at 25 °C. Relative rate of hemolytic activity was determined as the ratio of maximal hemolytic rate in the presence and absence of D22, respectively.

Permeabilization Experiments

GUVs were mixed with the buffer (10 mM MES, 150 mM NaCl, pH 6.5, or 20 mM Tris, 150 mM NaCl, pH 8.0), fluorescent dextran of 10 kDa in size (Sigma; FD10, final concentration of 1 mg/mL) and proteins in the following combinations: 50 nM Y406A alone, 5 μM D22M alone or premixed 50 nM Y406A + 5 μM D22M. Mixtures were incubated for 30 min at room temperature before imaging. Images were recorded on DMI6000 CS inverted microscope with TCS SP5 laser scanning system (both Leica Microsystems, Germany) with a 63 × oil-immersion objective (numerical aperture = 1.4). The rhodamine-containing GUV membrane was excited at 550 nm, and emission was detected from 570 to 600 nm. FD10 were excited at 488 nm, and emission was detected from 497 to 527 nm. Percent of permeabilization was calculated from green channel fluorescent intensities in ImageJ software, namely fluorescent intensities inside the vesicles were divided by background intensities outside the vesicles. For each condition 50 to 500 GUVs were analyzed.

Calcein Release from SUVs and LUVs

POPC:Chol 3:2 (mol:mol) lipids were used to prepare SUVs and POPC:Chol 1:1 (mol:mol) lipids for LUVs, both in 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 80 mM calcein, pH 8.0. Excess calcein was removed from vesicles suspension by gravity gel filtration on the Sephadex G-50 matrix (GE Healthcare, United Kingdom). Concentration of POPC and cholesterol was enzymatically determined with Phospholipids C kit and Free Cholesterol E kit (Wako Diagnostics, United States), respectively. Size and uniformity of SUVs and LUVs were checked with Dynamic Light Scattering (Zetasizer Nano, Malvern Panalytical, United Kingdom). We followed calcein release from SUVs in 96-well nontransparent microtiter plates (Costar, United States) with microplate reader Synergy MX (Biotek, United States). Vesicles with calcein were complemented with various combinations of 1 μM LLO or Y406A, and 5 μM D22 in buffer containing 10 mM HEPES, 150 mM NaCl, 1 mM EDTA at pH 6.5 or 8.0. From obtained fluorescence signals, we subtracted the fluorescence of the background, containing only vesicles with calcein and D22. Total release of calcein from SUVs was obtained by adding 2 mM detergent Triton X-100. To follow calcein release from LUVs, we used fluorimeter LS 55 Fluorescence Spectrometer (PerkinElmer, United States), measuring in a cuvette with final volume of 1 mL. Final concentration of lipids was 50 μM. Baseline fluorescence was followed for 300 s before the addition of proteins Y406A (30 nM final concentration) or D22 (1 μM final concentration). For the Y406A protein activation, pH was lowered to pH 6.5 by addition of 3 μL 7% HCl. For the Y406A protein inhibition, D22 was used either by immediate addition (10 s) after activation of Y406A or by preincubation of D22 and Y406A before or after addition to LUVs before or after lowering the pH. Fluorescence was then followed for at least 400 s before the addition of Triton X-100 (final concentration of 2 mM) to achieve full release of calcein.

Conjugation and Cleavage of D22M from MLVs

POPC:Chol:DSPE-PEG2000Mal 60:38:2 (molar ratio) and POPC:Chol:DSPE-PEG2000PDP 60:38:2 (molar ratio) mixtures were used to prepare MLVs in 20 mM HEPES, 300 mM NaCl, pH 8.0. D22M was conjugated on MLVs via maleimide conjugation (for MMP-9 cleavage) or PDP (for cleavage with the reducing agent) at 25 °C and shaking at 500 rpm under argon atmosphere overnight. Excess D22M was removed by centrifugation (800g, 8 min, 20 °C) and addition of buffer. First, the cleavage success of D22M from MLVs was analyzed by addition of 0.1 μg MMP-9 or 10 mM TCEP and incubation at 25 °C for 30 min. Afterward, the sample was centrifuged, and supernatant and pellet were analyzed by SDS-PAGE. Second, the binding of Y406A to D22M conjugated-MLVs and additional cleavage was analyzed. For this experiment, 1 μg of Y406A was added to MLVs with previously conjugated D22M and incubated for 30 min at 25 °C. Then the sample was centrifuged, the supernatant was saved for SDS-PAGE analysis, the pellet was resuspended in the buffer, and 0.1 μg MMP-9 or 10 mM TCEP were added and incubated for 30 min at 25 °C. The sample was centrifuged, and the supernatant and pellet were analyzed by SDS-PAGE.

Calcein Release from MLVs with Conjugated D22M

Calcein release was followed by using 96 well microtiter plates (Costar, United States). Here, DSPE-PEG2000Mal 60:38:2 MLVs conjugated with D22M were used for the cleavage experiments of D22M with MMP-9 and DSPE-PEG2000PDP 60:38:2 MLVs were used for the cleavage experiments of D22M with reducing agent TCEP. Final volume of reactions was 50 μL. First, the background fluorescence of 0.5 mM MLVs and buffer 20 mM HEPES, 300 mM NaCl, pH 8.0 (in the case of experiments with MMP-9 buffer also contained 10 mM CaCl2, 100 μM ZnSO4) was measured. Then Y406A was added at a final 500 nM concentration to the MLVs, and incubated for 30 min at 25 °C and 600 rpm. Afterward, 0.1 μg MMP-9 or 10 mM TCEP was added (incubated at 25 °C and 600 rpm for 30 min). Finally, 0.5 μL 3.5% HCl was added (incubated at 25 °C and 600 rpm for 30 min). Triton X-100 was added to achieve full release of the calcein. Fluorescence signal was measured by the Synergy MX microplate reader (Biotek, United States).

Statistical Analysis

For all experiments, raw data points are presented as circles on graphs, together with mean values ± standard deviation (SD). Unpaired two-tailed t tests assuming equal variances were performed using Origin 8.1 to present the differences between samples. Equalities of variances were assessed by two-sample F-test. A significant difference was determined by P-value <0.05. All the experiments were replicated at least three times and represented biological replicates.

Author Information

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  • Corresponding Author
  • Authors
    • Neža Omersa - Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia;  Biomedicine Doctoral Program, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
    • Saša Aden - Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia;  Biomedicine Doctoral Program, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
    • Matic Kisovec - Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia;  Orcidhttp://orcid.org/0000-0003-4131-2177
    • Marjetka Podobnik - Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia
  • Author Contributions

    N.O. and S.A. equally contributed to the work presented in this paper. N.O., S.A., M.P., and G.A. designed the study and experiments, N.O. and S.A. performed all experiments except SAXS, M.K. and M.P. performed SAXS experiments, N.O. and G.A. wrote the manuscript. All authors commented and approved the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The work presented in this paper was supported by The Slovenian Research Agency Grants P1-0391 and J4-8225. We thank Andreas Plückthun for the pRDV vector. We thank Alexey Kikhney from EMBL c/o DESY Synchrotron, Germany, to assist us with the SAXS experiments, Proposal No. SAXS-429.

References

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    Yasuga, Hiroki; Kawano, Ryuji; Takinoue, Masahiro; Tsuji, Yutaro; Osaki, Toshihisa; Kamiya, Koki; Miki, Norihisa; Takeuchi, Shoji
    PLoS One (2016), 11 (2), e0149667/1-e0149667/13CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Logical operations using biol. mols., such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biol. nanopores with DNA mols. for development of a logical operating system. We configured outputs "1" and "0" as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected elec. A neg.-AND (NAND) operation was successfully conducted within approx. 10 min, which is rapid compared with previous studies using unlabeled DNA. In addn., this operation was executed in a four-droplet network. DNA mols. and assocd. information were transferred among droplets via biol. nanopores. This system would facilitate linking of mols. and electronic interfaces. Thus, it could be applied to mol. robotics, genetic engineering, and even medical diagnosis and treatment.
  9. 9
    Faulhammer, D., Cukras, A. R., Lipton, R. J., and Landweber, L. F. (2000) Molecular computation: RNA solutions to chess problems. Proc. Natl. Acad. Sci. U. S. A. 97, 1385– 1389,  DOI: 10.1073/pnas.97.4.1385
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    Molecular computation: RNA solutions to chess problems
    Faulhammer, Dirk; Cukras, Anthony R.; Lipton, Richard J.; Landweber, Laura F.
    Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (4), 1385-1389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We have expanded the field of "DNA computers" to RNA and present a general approach for the soln. of satisfiability problems. As an example, we consider a variant of the "Knight problem," which asks generally what configurations of knights can one place on an n × n chess board such that no knight is attacking any other knight on the board. Using specific RNase digestion to manipulate strands of a 10-bit binary RNA library, we developed a mol. algorithm and applied it to a 3×3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine "bits" or placeholders in a combinatorial RNA library. We recovered a set of "winning" mols. that describe solns. to this problem.
  10. 10
    Choi, J. H. and Ostermeier, M. (2015) Rational design of a fusion protein to exhibit disulfide-mediated logic gate behavior. ACS Synth. Biol. 4, 400– 406,  DOI: 10.1021/sb500254g
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    Rational Design of a Fusion Protein to Exhibit Disulfide-Mediated Logic Gate Behavior
    Choi, Jay H.; Ostermeier, Marc
    ACS Synthetic Biology (2015), 4 (4), 400-406CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Synthetic cellular logic gates are primarily built from gene circuits owing to their inherent modularity. Single proteins can also possess logic gate functions and offer the potential to be simpler, quicker, and less dependent on cellular resources than gene circuits. However, the design of protein logic gates that are modular and integrate with other cellular components is a considerable challenge. As a step toward addressing this challenge, we describe the design, construction, and characterization of AND, ORN, and YES logic gates built by introducing disulfide bonds into RG13, a fusion of maltose binding protein and TEM-1 β-lactamase for which maltose is an allosteric activator of enzyme activity. We rationally designed these disulfide bonds to manipulate RG13's allosteric regulation mechanism such that the gating had maltose and reducing agents as input signals, and the gates could be toggled between different gating functions using redox agents, although some gates performed suboptimally.
  11. 11
    Fink, T., Lonzarić, J., Praznik, A., Plaper, T., Merljak, E., Leben, K., Jerala, N., Lebar, T., Strmšek, Ž., Lapenta, F., Benčina, M., and Jerala, R. (2019) Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat. Chem. Biol. 15, 115– 122,  DOI: 10.1038/s41589-018-0181-6
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    Design of fast proteolysis-based signaling and logic circuits in mammalian cells
    Fink, Tina; Lonzaric, Jan; Praznik, Arne; Plaper, Tjasa; Merljak, Estera; Leben, Katja; Jerala, Nina; Lebar, Tina; Strmsek, Ziga; Lapenta, Fabio; Bencina, Mojca; Jerala, Roman
    Nature Chemical Biology (2019), 15 (2), 115-122CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, the authors present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chem. or biol. signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.
  12. 12
    Moseley, F., Halámek, J., Kramer, F., Poghossian, A., Schöning, M. J., and Katz, E. (2014) An enzyme-based reversible CNOT logic gate realized in a flow system. Analyst 139, 1839– 1842,  DOI: 10.1039/c4an00133h
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    An enzyme-based reversible CNOT logic gate realized in a flow system
    Moseley, Fiona; Halamek, Jan; Kramer, Friederike; Poghossian, Arshak; Schoening, Michael J.; Katz, Evgeny
    Analyst (Cambridge, United Kingdom) (2014), 139 (8), 1839-1842CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD+ and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomol. realization of a CNOT gate is promising for integration into complex biomol. networks and future biosensor/biomedical applications.
  13. 13
    Stein, V. and Alexandrov, K. (2014) Protease-based synthetic sensing and signal amplification. Proc. Natl. Acad. Sci. U. S. A. 111, 15934– 15939,  DOI: 10.1073/pnas.1405220111
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    Protease-based synthetic sensing and signal amplification
    Stein, Viktor; Alexandrov, Kirill
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (45), 15934-15939CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The bottom-up design of protein-based signaling networks is a key goal of synthetic biol.; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based mol. switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously obsd. in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify mol. queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biol. process.
  14. 14
    Zhou, J., Arugula, M. A., Halámek, J., Pita, M., and Katz, E. (2009) Enzyme-based NAND and NOR logic gates with modular design. J. Phys. Chem. B 113, 16065– 16070,  DOI: 10.1021/jp9079052
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    14
    Enzyme-Based NAND and NOR Logic Gates with Modular Design
    Zhou, Jian; Arugula, Mary A.; Halamek, Jan; Pita, Marcos; Katz, Evgeny
    Journal of Physical Chemistry B (2009), 113 (49), 16065-16070CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    The logic gates NAND/NOR were mimicked by enzyme biocatalyzed reactions activated by sucrose, maltose and phosphate. The subunits performing AND/OR Boolean logic operations were designed using maltose phosphorylase and cooperative work of invertase/amyloglucosidase, resp. Glucose produced as the output signal from the AND/OR subunits was applied as the input signal for the INVERTER gate composed of alc. dehydrogenase, glucose oxidase, microperoxidase-11, ethanol and NAD+, which generated the final output in the form of NADH inverting the logic signal from 0 to 1 or from 1 to 0. The final output signal was amplified by a self-promoting biocatalytic system. In order to fulfill the Boolean properties of associativity and commutativity in logic networks, the final NADH output signal was converted to the initial signals of maltose and phosphate, thus allowing assembling of the same std. units in concatenated sequences. The designed modular approach, signal amplification and conversion processes open the way toward complex logic networks composed of std. elements resembling electronic integrated circuitries.
  15. 15
    Gao, X. J., Chong, L. S., Kim, M. S., and Elowitz, M. B. (2018) Programmable protein circuits in living cells. Science 361, 1252– 1258,  DOI: 10.1126/science.aat5062
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    Programmable protein circuits in living cells
    Gao, Xiaojing J.; Chong, Lucy S.; Kim, Matthew S.; Elowitz, Michael B.
    Science (Washington, DC, United States) (2018), 361 (6408), 1252-1258CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnol. applications.
  16. 16
    Hilburger, C. E., Jacobs, M. L., Lewis, K. R., Peruzzi, J. A., and Kamat, N. P. (2019) Controlling Secretion in Artificial Cells with a Membrane and Gate. ACS Synth. Biol. 8, 1224– 1230,  DOI: 10.1021/acssynbio.8b00435
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    16
    Controlling Secretion in Artificial Cells with a Membrane AND Gate
    Hilburger, Claire E.; Jacobs, Miranda L.; Lewis, Kamryn R.; Peruzzi, Justin A.; Kamat, Neha P.
    ACS Synthetic Biology (2019), 8 (6), 1224-1230CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The assembly of channel proteins into vesicle membranes is a useful strategy to control activities of vesicle-based systems. Here, the authors developed a membrane AND gate that responds to both a fatty acid and a pore-forming channel protein to induce the release of encapsulated cargo. The authors explored how membrane compn. affects the functional assembly of α-hemolysin into phospholipid vesicles as a function of oleic acid content and α-hemolysin concn. The authors then showed that the authors could induce α-hemolysin assembly when the authors added oleic acid micelles to a specific compn. of phospholipid vesicles. Finally, the authors demonstrated that the membrane AND gate could be coupled to a gene expression system. The study provides a new method to control the temporal dynamics of vesicle permeability by controlling when the functional assembly of a channel protein into synthetic vesicles occurs. Furthermore, a membrane AND gate that utilizes membrane-assocg. biomols. introduces a new way to implement Boolean logic that should complement genetic logic circuits and ultimately enhance the capabilities of artificial cellular systems.
  17. 17
    Adamala, K. P., Martin-Alarcon, D. A., Guthrie-Honea, K. R., and Boyden, E. S. (2017) Engineering genetic circuit interactions within and between synthetic minimal cells. Nat. Chem. 9, 431– 439,  DOI: 10.1038/nchem.2644
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    17
    Engineering genetic circuit interactions within and between synthetic minimal cells
    Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
    Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
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    Einfalt, T., Goers, R., Dinu, I. A., Najer, A., Spulber, M., Onaca-Fischer, O., and Palivan, C. G. (2015) Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes. Nano Lett. 15, 7596– 7603,  DOI: 10.1021/acs.nanolett.5b03386
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    18
    Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes
    Einfalt, Tomaz; Goers, Roland; Dinu, Ionel Adrian; Najer, Adrian; Spulber, Mariana; Onaca-Fischer, Ozana; Palivan, Cornelia G.
    Nano Letters (2015), 15 (11), 7596-7603CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    The development of advanced stimuli-responsive systems for medicine, catalysis, or technol. requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to mol. transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein "gates" in the membrane. The outer membrane protein F (OmpF) porin was chem. modified with a pH-responsive mol. cap to serve as "gate" producing pH-driven mol. flow through the membrane and control the in situ enzymic activity. This strategy provides complex reaction spaces necessary in "smart" medicine and for biomimetic engineering of artificial cells.
  19. 19
    Einfalt, T., Witzigmann, D., Edlinger, C., Sieber, S., Goers, R., Najer, A., Spulber, M., Onaca-Fischer, O., Huwyler, J., and Palivan, C. G. (2018) Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment. Nat. Commun. 9, 1127,  DOI: 10.1038/s41467-018-03560-x
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    19
    Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment
    Einfalt T; Edlinger C; Goers R; Najer A; Spulber M; Onaca-Fischer O; Palivan C G; Einfalt T; Witzigmann D; Sieber S; Huwyler J; Goers R
    Nature communications (2018), 9 (1), 1127 ISSN:.
    Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.
  20. 20
    van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008) Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112– 124,  DOI: 10.1038/nrm2330
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    Membrane lipids: where they are and how they behave
    van Meer, Gerrit; Voelker, Dennis R.; Feigenson, Gerald W.
    Nature Reviews Molecular Cell Biology (2008), 9 (2), 112-124CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. Throughout the biol. world, a 30 Å hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochem. and biophys. findings have provided a detailed model of the compn. and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases, and transporters, plus the intrinsic phys. phase behavior of lipids and their interactions with membrane proteins, to create the unique compns. and multiple functionalities of their individual membranes.
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    Golynskiy, M. V., Koay, M. S., Vinkenborg, J. L., and Merkx, M. (2011) Engineering protein switches: sensors, regulators, and spare parts for biology and biotechnology. ChemBioChem 12, 353– 361,  DOI: 10.1002/cbic.201000642
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    Engineering Protein Switches: Sensors, Regulators, and Spare Parts for Biology and Biotechnology
    Golynskiy, Misha V.; Koay, Melissa S.; Vinkenborg, Jan L.; Merkx, Maarten
    ChemBioChem (2011), 12 (3), 353-361CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The authors review the underlying engineering concepts for constructing protein switches, most of which are inspired by or reminiscent of natural examples. The authors focus on those strategies that have the potential to be generally applicable, and discuss them according to their mechanism of action in order of increasing modularity and decreasing domain integration. Finally, the authors identify the opportunities and challenges assocd. with these different engineering concepts and discuss future perspectives.
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    Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L., and Beltram, F. (2010) Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat. Methods 7, 516– 518,  DOI: 10.1038/nmeth.1471
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    Simultaneous intracellular chloride and pH measurements using a GFP-based sensor
    Arosio, Daniele; Ricci, Fernanda; Marchetti, Laura; Gualdani, Roberta; Albertazzi, Lorenzo; Beltram, Fabio
    Nature Methods (2010), 7 (7), 516-518CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concn. in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.
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    Dal Peraro, M. and van der Goot, F. G. (2016) Pore-forming toxins: ancient, but never really out of fashion. Nat. Rev. Microbiol. 14, 77– 92,  DOI: 10.1038/nrmicro.2015.3
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    Pore-forming toxins: ancient, but never really out of fashion
    Dal Peraro Matteo; van der Goot F Gisou
    Nature reviews. Microbiology (2016), 14 (2), 77-92 ISSN:.
    Pore-forming toxins (PFTs) are virulence factors produced by many pathogenic bacteria and have long fascinated structural biologists, microbiologists and immunologists. Interestingly, pore-forming proteins with remarkably similar structures to PFTs are found in vertebrates and constitute part of their immune system. Recently, structural studies of several PFTs have provided important mechanistic insights into the metamorphosis of PFTs from soluble inactive monomers to cytolytic transmembrane assemblies. In this Review, we discuss the diverse pore architectures and membrane insertion mechanisms that have been revealed by these studies, and we consider how these features contribute to binding specificity for different membrane targets. Finally, we explore the potential of these structural insights to enable the development of novel therapeutic strategies that would prevent both the establishment of bacterial resistance and an excessive immune response.
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    Anderluh, G. and Lakey, J. H. (2008) Disparate proteins use similar architectures to damage membranes. Trends Biochem. Sci. 33, 482– 490,  DOI: 10.1016/j.tibs.2008.07.004
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    Disparate proteins use similar architectures to damage membranes
    Anderluh, Gregor; Lakey, Jeremy H.
    Trends in Biochemical Sciences (2008), 33 (10), 482-490CODEN: TBSCDB; ISSN:0968-0004. (Elsevier B.V.)
    A review. Membrane disruption can efficiently alter cellular function; indeed, pore-forming toxins (PFTs) are well known as important bacterial virulence factors. However, recent data have revealed that structures similar to those found in PFTs are found in membrane active proteins across disparate phyla. Many similarities can be identified only at the 3D-structural level. Of note, domains found in membrane-attack complex proteins of complement and perforin (MACPF) resemble cholesterol-dependent cytolysins from Gram-pos. bacteria, and the Bcl family of apoptosis regulators share similar architectures with Escherichia coli pore-forming colicins. These and other correlations provide considerable help in understanding the structural requirements for membrane binding and pore formation.
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    Laventie, B., Potrich, C., Atmanène, C., Saleh, M., Joubert, O., Viero, G., Bachmeyer, C., Antonini, V., Mancini, I., Cianferani-Sanglier, S., Keller, D., Colin, D. A., Bourcier, T., Anderluh, G., van Dorsselaer, A., Dalla Serra, M., and Prévost, G. (2013) p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions. Biochem. J. 450, 559– 571,  DOI: 10.1042/BJ20121628
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    25
    p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions
    Laventie, Benoit-Joseph; Potrich, Cristina; Atmanene, Cedric; Saleh, Maher; Joubert, Olivier; Viero, Gabriella; Bachmeyer, Christoph; Antonini, Valeria; Mancini, Ines; Cianferani-Sanglier, Sarah; Keller, Daniel; Colin, Didier A.; Bourcier, Tristan; Anderluh, Gregor; van Dorsselaer, Alain; Dalla Serra, Mauro; Prevost, Gilles
    Biochemical Journal (2013), 450 (3), 559-571CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus β-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chems. have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 μM for Ca2+ activation and of 2-50 μM for cell lysis. SCns were obsd. to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramol. interactions (1:1 stoichiometry) between SCns and class S proteins in soln., but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concns. according to the no. of benzenes (30-300 μM; SC8>SC6»SC4). The inhibitory properties of SCns were also obsd. in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.
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    Booth, M. J., Schild, V. R., Graham, A. D., Olof, S. N., and Bayley, H. (2016) Light-activated communication in synthetic tissues. Sci. Adv. 2, e1600056  DOI: 10.1126/sciadv.1600056
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    Light-activated communication in synthetic tissues
    Booth, Michael J.; Schild, Vanessa Restrepo; Graham, Alexander D.; Olof, Sam N.; Bayley, Hagan
    Science Advances (2016), 2 (4), e1600056/1-e1600056/11CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)
    We have previously used three-dimensional (3D) printing to prep. tissue-like materials in which picoliter aq. compartments are sepd. by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional elec. communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way.
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    Provoda, C. J., Stier, E. M., and Lee, K. D. (2003) Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin. J. Biol. Chem. 278, 35102– 35108,  DOI: 10.1074/jbc.M305411200
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    Tumor cell killing enabled by Listeriolysin O-liposome-mediated delivery of the protein toxin gelonin
    Provoda, Chester J.; Stier, Ethan M.; Lee, Kyung-Dall
    Journal of Biological Chemistry (2003), 278 (37), 35102-35108CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromol., however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degrdn. within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro expts., co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of ∼0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equiv. concns. of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into s.c. solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes contg. both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.
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    Robertson, J. W. F., Rodrigues, C. G., Stanford, V. M., Rubinson, K. A., Krasilnikov, O. V., and Kasianowicz, J. J. (2007) Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl. Acad. Sci. U. S. A. 104, 8207– 8211,  DOI: 10.1073/pnas.0611085104
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    Single-molecule mass spectrometry in solution using a solitary nanopore
    Robertson, Joseph W. F.; Rodrigues, Claudio G.; Stanford, Vincent M.; Rubinson, Kenneth A.; Krasilnikov, Oleg V.; Kasianowicz, John J.
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (20), 8207-8211CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The authors introduce a 2-dimensional method for mass spectrometry in soln. that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) mols. that enter a single α-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of mols. in soln.
  29. 29
    Clarke, J., Wu, H., Jayasinghe, L., Patel, A., Reid, S., and Bayley, H. (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265– 270,  DOI: 10.1038/nnano.2009.12
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    Continuous base identification for single-molecule nanopore DNA sequencing
    Clarke, James; Wu, Hai-Chen; Jayasinghe, Lakmal; Patel, Alpesh; Reid, Stuart; Bayley, Hagan
    Nature Nanotechnology (2009), 4 (4), 265-270CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    A single-mol. method for sequencing DNA that does not require fluorescent labeling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide mols. from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, the authors show that a protein nanopore with a covalently attached adapter mol. can continuously identify unlabeled nucleoside 5'-monophosphate mols. with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four std. DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analyzing epigenetic modifications.
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    Wang, Y., Montana, V., Grubišić, V., Stout, R. F., Parpura, V., and Gu, L. Q. (2015) Nanopore sensing of botulinum toxin type B by discriminating an enzymatically cleaved peptide from a synaptic protein synaptobrevin 2 derivative. ACS Appl. Mater. Interfaces 7, 184– 192,  DOI: 10.1021/am5056596
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    Nanopore Sensing of Botulinum Toxin Type B by Discriminating an Enzymatically Cleaved Peptide from a Synaptic Protein Synaptobrevin 2 Derivative
    Wang, Yong; Montana, Vedrana; Grubisic, Vladimir; Stout, Randy F.; Parpura, Vladimir; Gu, Li-Qun
    ACS Applied Materials & Interfaces (2015), 7 (1), 184-192CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and assocd. with legal and ethical constrains. Various biochem., optical and mech. methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin's enzymic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 deriv. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concn. was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophys. mechanisms for biopolymer transportation.
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    Wang, Y., Zheng, D., Tan, Q., Wang, M., and Gu, L. (2011) Nanopore-based detection of circulating microRNAs in lung cancer patients. Nat. Nanotechnol. 6, 668– 674,  DOI: 10.1038/nnano.2011.147
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    Nanopore-based detection of circulating microRNAs in lung cancer patients
    Wang, Yong; Zheng, Da-Li; Tan, Qiu-Lin; Wang, Michael X.; Gu, Li-Qun
    Nature Nanotechnology (2011), 6 (10), 668-674CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    MicroRNAs are short RNA mols. that regulate gene expression, and have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, detecting microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the α-haemolysin protein can selectively detect microRNAs at the single mol. level in plasma samples from lung cancer patients without the need for labels or amplification of the microRNA. The sensor, which uses a programmable oligonucleotide probe to generate a target-specific signature signal, can quantify subpicomolar levels of cancer-assocd. microRNAs and can distinguish single-nucleotide differences between microRNA family members. This approach is potentially useful for quant. microRNA detection, the discovery of disease markers and non-invasive early diagnosis of cancer.
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    Braha, O., Bayley, H., Gu, L. Q., Zhou, L., Lu, X., and Cheley, S. (2000) Simultaneous stochastic sensing of divalent metal ions. Nat. Biotechnol. 18, 1005– 1007,  DOI: 10.1038/79275
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    Simultaneous stochastic sensing of divalent metal ions
    Braha, Orit; Gu, Lin-Qun; Zhou, Li; Lu, Xiaofeng; Cheley, Stephen; Bayley, Hagan
    Nature Biotechnology (2000), 18 (9), 1005-1007CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    Stochastic sensing is an emerging anal. technique that relies upon single-mol. detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concn. of an analyte and, through a characteristic signature, its identity. Here, we show that the concns. of two or more divalent metal ions in soln. can be detd. simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.
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    Villar, G., Graham, A. D., and Bayley, H. (2013) A tissue-like printed material. Science 340, 48– 52,  DOI: 10.1126/science.1229495
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    A Tissue-Like Printed Material
    Villar, Gabriel; Graham, Alexander D.; Bayley, Hagan
    Science (Washington, DC, United States) (2013), 340 (6128), 48-52CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Living cells communicate and cooperate to produce the emergent properties of tissues. Synthetic mimics of cells, such as liposomes, are typically incapable of cooperation and therefore cannot readily display sophisticated collective behavior. We printed tens of thousands of picoliter aq. droplets that become joined by single lipid bilayers to form a cohesive material with cooperating compartments. Three-dimensional structures can be built with heterologous droplets in software-defined arrangements. The droplet networks can be functionalized with membrane proteins; for example, to allow rapid elec. communication along a specific path. The networks can also be programmed by osmolarity gradients to fold into otherwise unattainable designed structures. Printed droplet networks might be interfaced with tissues, used as tissue engineering substrates, or developed as mimics of living tissue.
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    Elani, Y., Law, R. V., and Ces, O. (2014) Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 5, 5305,  DOI: 10.1038/ncomms6305
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    Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways
    Elani, Yuval; Law, Robert V.; Ces, Oscar
    Nature Communications (2014), 5 (), 5305CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    In the discipline of bottom-up synthetic biol., vesicles define the boundaries of artificial cells and are increasingly being used as biochem. microreactors operating in physiol. environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chem. pathway to be sepd. in space, this platform bridges the gap between table-top chem. and chem. that is performed within vesicles.
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    Hamon, M. A., Ribet, D., Stavru, F., and Cossart, P. (2012) Listeriolysin O: the Swiss army knife of Listeria. Trends Microbiol. 20, 360– 368,  DOI: 10.1016/j.tim.2012.04.006
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    Listeriolysin O: the Swiss army knife of Listeria
    Hamon, Melanie Anne; Ribet, David; Stavru, Fabrizia; Cossart, Pascale
    Trends in Microbiology (2012), 20 (8), 360-368CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
    A review. Listeriolysin O (LLO) is a toxin produced by Listeria monocytogenes, an opportunistic bacterial pathogen responsible for the disease listeriosis. This disease starts with the ingestion of contaminated foods and mainly affects immunocompromised individuals, newborns, and pregnant women. In the lab., L. monocytogenes is used as a model organism to study processes such as cell invasion, intracellular survival, and cell-to-cell spreading, as this Gram-pos. bacterium has evolved elaborate mol. strategies to subvert host cell functions. LLO is a major virulence factor originally shown to be crucial for bacterial escape from the internalization vacuole after entry into cells. However, recent studies are revisiting the role of LLO during infection and are revealing new insights into the action of LLO, in particular before bacterial entry. These latest findings along with their impact on the infectious process will be discussed.
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    Köster, S., van Pee, K., Hudel, M., Leustik, M., Rhinow, D., Kühlbrandt, W., Chakraborty, T., and Yildiz, O. (2014) Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation. Nat. Commun. 5, 3690,  DOI: 10.1038/ncomms4690
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    Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation
    Koster Stefan; van Pee Katharina; Rhinow Daniel; Kuhlbrandt Werner; Yildiz Ozkan; Hudel Martina; Leustik Martin; Chakraborty Trinad
    Nature communications (2014), 5 (), 3690 ISSN:.
    Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca(2+) oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca(2+) uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.
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    Ruan, Y., Rezelj, S., Bedina Zavec, A., Anderluh, G., and Scheuring, S. (2016) Listeriolysin O membrane damaging activity involves arc formation and lineaction - implication for Listeria monocytogenes escape from phagocytic vacuole. PLoS Pathog. 12, e1005597  DOI: 10.1371/journal.ppat.1005597
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    Listeriolysin O membrane damaging activity involves arc formation and lineaction - implication for Listeria monocytogenes escape from phagocytic vacuole
    Ruan, Yi; Rezelj, Sasa; Zavec, Apolonija Bedina; Anderluh, Gregor; Scheuring, Simon
    PLoS Pathogens (2016), 12 (4), e1005597/1-e1005597/18CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
    Listeriolysin-O (LLO) plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood, probably due to unavailability of suitable exptl. tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed at. force microscopy (HS-AFM) featuring high spatio-temporal resoln. on model membranes and optical microscopy on giant unilamellar vesicles (GUVs) to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concn. and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context.
  38. 38
    Podobnik, M., Marchioretto, M., Zanetti, M., Bavdek, A., Kisovec, M., Cajnko, M. M., Lunelli, L., Serra, M. D., and Anderluh, G. (2015) Plasticity of lysteriolysin O pores and its regulation by pH and unique histidine. Sci. Rep. 5, 9623,  DOI: 10.1038/srep09623
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    Plasticity of Listeriolysin O Pores and its Regulation by pH and Unique Histidine
    Podobnik, Marjetka; Marchioretto, Marta; Zanetti, Manuela; Bavdek, Andrej; Kisovec, Matic; Cajnko, Misa Mojca; Lunelli, Lorenzo; Dalla Serra, Mauro; Anderluh, Gregor
    Scientific Reports (2015), 5 (), 9623/1-9623/10CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irresp. of pH. While pore diams. at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.
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    Mulvihill, E., Van Pee, K., Mari, S. A., Müller, D. J., and Yildiz, Ö. (2015) Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O. Nano Lett. 15, 6965– 6973,  DOI: 10.1021/acs.nanolett.5b02963
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    Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O
    Mulvihill Estefania; Mari Stefania A; Muller Daniel J; van Pee Katharina; Yildiz Ozkan
    Nano letters (2015), 15 (10), 6965-73 ISSN:.
    Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.
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    Kisovec, M., Rezelj, S., Knap, P., Cajnko, M., Caserman, S., Flašker, A., Žnidaršič, N., Repič, M., Mavri, J., Ruan, Y., Scheuring, S., Podobnik, M., and Anderluh, G. (2017) Engineering a pH responsive pore forming protein. Sci. Rep. 7, 42231,  DOI: 10.1038/srep42231
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    Engineering a pH responsive pore forming protein
    Kisovec, Matic; Rezelj, Sasa; Knap, Primoz; Cajnko, Misa Mojca; Caserman, Simon; Flasker, Ajda; Znidarsic, Nada; Repic, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor
    Scientific Reports (2017), 7 (), 42231CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural anal. coupled with mol. dynamics and mutational anal., we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Mol. dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnol. applications such as controlled release of substances or as a sensor of environmental pH.
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    Dreier, B., and Plückthun, A. (2011) Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries, in PCR Protocols. Methods in Molecular Biology (Methods and Protocols), (Park, D. J., Ed.), pp 283– 306, Humana Press, Totowa, NJ.
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    Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A. (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575– 82,  DOI: 10.1038/nbt962
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    High-affinity binders selected from designed ankyrin repeat protein libraries
    Binz, H. Kaspar; Amstutz, Patrick; Kohl, Andreas; Stumpp, Michael T.; Briand, Christophe; Forrer, Patrik; Gruetter, Markus G.; Plueckthun, Andreas
    Nature Biotechnology (2004), 22 (5), 575-582CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat nos. with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and detd. the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 Å resoln. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding mols. and, because of the very favorable biophys. properties of the designed AR proteins, an attractive alternative to antibody libraries.
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    Bavdek, A., Gekara, N. O., Priselac, D., Gutiérrez Aguirre, I., Darji, A., Chakraborty, T., Macek, P., Lakey, J. H., Weiss, S., and Anderluh, G. (2007) Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O. Biochemistry 46, 4425– 4437,  DOI: 10.1021/bi602497g
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    Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O
    Bavdek, Andrej; Gekara, Nelson O.; Priselac, Dragan; Gutierrez Aguirre, Ion; Darji, Ayub; Chakraborty, Trinad; Macek, Peter; Lakey, Jeremy H.; Weiss, Siegfried; Anderluh, Gregor
    Biochemistry (2007), 46 (14), 4425-4437CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concns. in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiol. or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approx. 35 mol % cholesterol, a concn. range typically found in lipid rafts. The narrow transition region of cholesterol concn. sepg. low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is detd. by the most exposed 3β-hydroxy group of cholesterol. In addn., LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiol. or slightly basic pH values. The results clearly show that LLO activity at physiol. pH cannot be neglected and that its action at sites distal to cell entry may have important physiol. consequences for Listeria pathogenesis.
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    Sallee, N. A., Yeh, B. J., and Lim, W. A. (2007) Engineering modular protein interaction switches by sequence overlap. J. Am. Chem. Soc. 129, 4606– 4611,  DOI: 10.1021/ja0672728
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    Engineering Modular Protein Interaction Switches by Sequence Overlap
    Sallee, Nathan A.; Yeh, Brian J.; Lim, Wendell A.
    Journal of the American Chemical Society (2007), 129 (15), 4606-4611CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Many cellular signaling pathways contain proteins whose interactions change in response to upstream inputs, allowing for conditional activation or repression of the interaction based on the presence of the input mol. The ability to engineer similar regulation into protein interaction elements would provide us with powerful tools for controlling cell signaling. Here we describe an approach for engineering diverse synthetic protein interaction switches. Specifically, by overlapping the sequences of pairs of protein interaction domains and peptides, we have been able to generate mutually exclusive regulation over their interactions. Thus, the hybrid protein (which is composed of the two overlapped interaction modules) can bind to either of the two resp. ligands for those modules, but not to both simultaneously. We show that these synthetic switch proteins can be used to regulate specific protein-protein interactions in vivo. These switches allow us to disrupt an interaction with the addn. or activation of a protein input that has no natural connection to the interaction in question. Therefore, they give us the ability to make novel connections between normally unrelated signaling pathways and to rewire the input/output relationships of cellular behaviors. Our expts. also suggest a possible mechanism by which complex regulatory proteins might have evolved from simpler components.
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    Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., and Hahn, K. M. (2010) Engineered allosteric activation of kinases in living cells. Nat. Biotechnol. 28, 743– 747,  DOI: 10.1038/nbt.1639
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    Engineered allosteric activation of kinases in living cells
    Karginov, Andrei V.; Ding, Feng; Kota, Pradeep; Dokholyan, Nikolay V.; Hahn, Klaus M.
    Nature Biotechnology (2010), 28 (7), 743-747CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions. Catalytic activity is restored by addn. of rapamycin or non-immunosuppresive rapamycin analogs. Mol. modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.
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    Edwards, W. R., Busse, K., Allemann, R. K., and Jones, D. D. (2008) Linking the functions of unrelated proteins using a novel directed evolution domain insertion method. Nucleic Acids Res. 36, e78  DOI: 10.1093/nar/gkn363
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    Linking the functions of unrelated proteins using a novel directed evolution domain insertion method
    Edwards, Wayne R.; Busse, Kathy; Allemann, Rudolf K.; Jones, D. Dafydd
    Nucleic Acids Research (2008), 36 (13), e78/1-e78/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the sep. protein activities, thus providing a general strategy for constructing mol. switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were obsd. in loops, several also occurred close to the termini of α-helixes and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.
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    Ha, J. H., Butler, J. S., Mitrea, D. M., and Loh, S. N. (2006) Modular enzyme design: Regulation by mutually exclusive protein folding. J. Mol. Biol. 357, 1058– 1062,  DOI: 10.1016/j.jmb.2006.01.073
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    Modular Enzyme Design: Regulation by Mutually Exclusive Protein Folding
    Ha, Jeung-Hoi; Butler, James S.; Mitrea, Diana M.; Loh, Stewart N.
    Journal of Molecular Biology (2006), 357 (4), 1058-1062CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
    A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topol. constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodn. tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.
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    Tweten, R. K., Hotze, E. M., and Wade, K. R. (2015) The unique molecular choreography of giant pore formation by the cholesterol-dependent cytolysins of Gram-positive bacteria. Annu. Rev. Microbiol. 69, 323– 340,  DOI: 10.1146/annurev-micro-091014-104233
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    The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria
    Tweten, Rodney K.; Hotze, Eileen M.; Wade, Kristin R.
    Annual Review of Microbiology (2015), 69 (), 323-340CODEN: ARMIAZ; ISSN:0066-4227. (Annual Reviews)
    A review. The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophys., and biochem. analyses revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. The authors' knowledge of the mol. details of these dramatic structural changes in CDCs has transformed the understanding of how giant pore complexes are assembled and has been crit. to the understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs were employed in pore-independent processes.
  49. 49
    Song, L., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley, H., and Gouaux, J. E. (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859– 1866,  DOI: 10.1126/science.274.5294.1859
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    Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore
    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric
    Science (Washington, D. C.) (1996), 274 (5294), 1859-1866CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The structure of the Staphylococcus aureus α-hemolysin pore has been detd. to 1.9 Å resoln. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 Å in length, that runs along the sevenfold axis and ranges from 14 Å to 46 Å in diam. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each promoter contributes two β strands, each 65 Å long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 Å wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-sol. protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.
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    Podobnik, M., Savory, P., Rojko, N., Kisovec, M., Wood, N., Hambley, R., Pugh, J., Wallace, E. J., McNeill, L., Bruce, M., Liko, I., Allison, T. M., Mehmood, S., Yilmaz, N., Kobayashi, T., Gilbert, R. J. C., Robinson, C. V., Jayasinghe, L., and Anderluh, G. (2016) Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat. Commun. 7, 11598,  DOI: 10.1038/ncomms11598
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    Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly
    Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E. Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M.; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J. C.; Robinson, Carol V.; Jayasinghe, Lakmal; Anderluh, Gregor
    Nature Communications (2016), 7 (), 11598CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of addnl. luminal compartments as commonly found in other toxin pores. Mutagenic anal. and at. force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.
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    Tsuchikama, K. and An, Z. (2018) Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell 9, 33– 46,  DOI: 10.1007/s13238-016-0323-0
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    Antibody-drug conjugates: recent advances in conjugation and linker chemistries
    Tsuchikama, Kyoji; An, Zhiqiang
    Protein & Cell (2018), 9 (1), 33-46CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press)
    The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small mols. (payloads) through chem. linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based mol. platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris and Kadcyla, this drug class has been rapidly growing along with about 60 ADCs currently in clin. trials. In this article, we briefly review mol. aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clin. effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into mol. design and strategies for clin. effective ADCs from medicinal chem. and pharmacol. standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an esp. promising path to improving ADC design, which will open the way for novel cancer therapeutics.
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    Leriche, G., Chisholm, L., and Wagner, A. (2012) Cleavable linkers in chemical biology. Bioorg. Med. Chem. 20, 571– 582,  DOI: 10.1016/j.bmc.2011.07.048
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    Cleavable linkers in chemical biology
    Leriche, Geoffray; Chisholm, Louise; Wagner, Alain
    Bioorganic & Medicinal Chemistry (2012), 20 (2), 571-582CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    A review. Interest in cleavable linkers is growing due to the rapid development and expansion of chem. biol. The chem. constrains imposed by the biol. conditions cause significant challenges for org. chemists. In this review we will present an overview of the cleavable linkers used in chem. biol. classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradn., electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.
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    Cajnko, M. M., Marušić, M., Kisovec, M., Rojko, N., Benčina, M., Caserman, S., and Anderluh, G. (2015) Listeriolysin O affects the permeability of Caco-2 monolayer in a pore-dependent and Ca2+-independent manner. PLoS One 10, e0130471  DOI: 10.1371/journal.pone.0130471
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    Listeriolysin O affects the permeability of Caco-2 monolayer in a pore-dependent and Ca2+-independent manner
    Cajnko Misa, Mojca; Marusic, Maja; Kisovec, Matic; Rojko, Nejc; Bencina, Mojca; Caserman, Simon; Anderluh, Gregor
    PLoS One (2015), 10 (6), e0130471/1-e0130471/21CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Apical application of both toxins causes a significant drop in transepithelial elec. resistance (TEER), with higher LLO concns. or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and assocd. actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was obsd. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphol. and physiol. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concns. than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.
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    Gerweck, L. E. and Seetharaman, K. (1996) Cellular pH gradient in tumor versus normal tissue: Potential exploitation for the treatment of cancer. Cancer Res. 56, 1194– 1198
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    Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer
    Gerweck, Leo E.; Seetharaman, Kala
    Cancer Research (1996), 56 (6), 1194-8CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to primarily represent tissue extracellular pH) is substantially and consistently lower in tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy estd. that intracellular pH is essentially identical or slightly more basic in tumor compared to normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in tumor compared to normal tissue: in normal tissue the extracellular pH is relatively basic, and in tumor tissue the magnitude of the pH gradient is reduced or reversed. This difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized is strongly dependent on the pH of their milieu. Weakly acidic drugs which are relatively lipid sol. in their nonionized state may diffuse freely across the cell membrane and, upon entering a relatively basic intracellular compartment, become trapped and accumulate within a cell, leading to substantial differences in the intracellular/extracellular drug distribution between tumor and normal tissue for drugs exhibiting appropriate pKas.
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    Cerutti, P. A. (1985) Prooxidant states and tumor promotion. Science 227, 375– 381,  DOI: 10.1126/science.2981433
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    Prooxidant states and tumor promotion
    Cerutti, Peter A.
    Science (Washington, DC, United States) (1985), 227 (4685), 375-81CODEN: SCIEAS; ISSN:0036-8075.
    There is convincing evidence that cellular prooxidant states-i.e., increased concns. of active O and org. peroxides and radicals-can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric O, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P 450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low mol. wt. scavenger mols., and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.
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    Bergers, G., Brekken, R., McMahon, G., Vu, T. H., Itoh, T., Tamaki, K., Tanzawa, K., Thorpe, P., Itohara, S., Werb, Z., and Hanahan, D. (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2, 737– 744,  DOI: 10.1038/35036374
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    Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis
    Bergers, Gabriele; Brekken, Rolf; McMahon, Gerald; Vu, Thiennu H.; Itoh, Takeshi; Tamaki, Kazuhiko; Tanzawa, Kazuhiko; Thorpe, Philip; Itohara, Shigeyoshi; Werb, Zena; Hanahan, Douglas
    Nature Cell Biology (2000), 2 (10), 737-744CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
    During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumor growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumor no. and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumor growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.
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    Karimi, M., Ghasemi, A., Sahandi Zangabad, P., Rahighi, R., Moosavi Basri, S. M., Mirshekari, H., Amiri, M., Shafaei Pishabad, Z., Aslani, A., Bozorgomid, M., Ghosh, D., Beyzavi, A., Vaseghi, A., Aref, A. R., Haghani, L., Bahrami, S., and Hamblin, M. R. (2016) Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems. Chem. Soc. Rev. 45, 1457– 1501,  DOI: 10.1039/C5CS00798D
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    Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems
    Karimi, Mahdi; Ghasemi, Amir; Sahandi Zangabad, Parham; Rahighi, Reza; Moosavi Basri, S. Masoud; Mirshekari, H.; Amiri, M.; Shafaei Pishabad, Z.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A. R.; Haghani, L.; Bahrami, S.; Hamblin, Michael R.
    Chemical Society Reviews (2016), 45 (5), 1457-1501CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    New achievements in the realm of nanoscience and innovative techniques of nanomedicine have moved micro/nanoparticles (MNPs) to the point of becoming actually useful for practical applications in the near future. Various differences between the extracellular and intracellular environments of cancerous and normal cells and the particular characteristics of tumors such as physicochem. properties, neovasculature, elasticity, surface elec. charge, and pH have motivated the design and fabrication of inventive "smart" MNPs for stimulus-responsive controlled drug release. These novel MNPs can be tailored to be responsive to pH variations, redox potential, enzymic activation, thermal gradients, magnetic fields, light, and ultrasound (US), or can even be responsive to dual or multi-combinations of different stimuli. This unparalleled capability has increased their importance as site-specific controlled drug delivery systems (DDSs) and has encouraged their rapid development in recent years. An in-depth understanding of the underlying mechanisms of these DDS approaches is expected to further contribute to this groundbreaking field of nanomedicine. Smart nanocarriers in the form of MNPs that can be triggered by internal or external stimulus are summarized and discussed in the present review, including pH-sensitive peptides and polymers, redox-responsive micelles and nanogels, thermo- or magnetic-responsive nanoparticles (NPs), mech.- or elec.-responsive MNPs, light or ultrasound-sensitive particles, and multi-responsive MNPs including dual stimuli-sensitive nanosheets of graphene. This review highlights the recent advances of smart MNPs categorized according to their activation stimulus (phys., chem., or biol.) and looks forward to future pharmaceutical applications.
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    Zahnd, C., Amstutz, P., and Plückthun, A. (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269– 279,  DOI: 10.1038/nmeth1003
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    Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target
    Zahnd, Christian; Amstutz, Patrick; Plueckthun, Andreas
    Nature Methods (2007), 4 (3), 269-279CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Ribosome display is an in vitro selection and evolution technol. for proteins and peptides from large libraries. As it is performed entirely in vitro, there are two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the no. of ribosomes and different mRNA mols. present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations (Box 1). A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide (Fig. 1). The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for anal. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 ext. for in vitro translation, based on the work originally described and further refined in our lab. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display is also included in this issue.
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    Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., Ward, C. W., Joos, T. O., and Plückthun, A. (2007) A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J. Mol. Biol. 369, 1015– 28,  DOI: 10.1016/j.jmb.2007.03.028
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    A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2
    Zahnd, Christian; Wyler, Emanuel; Schwenk, Jochen M.; Steiner, Daniel; Lawrence, Michael C.; McKern, Neil M.; Pecorari, Frederic; Ward, Colin W.; Joos, Thomas O.; Plueckthun, Andreas
    Journal of Molecular Biology (2007), 369 (4), 1015-1028CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Designed ankyrin repeat proteins (DARPins) are a novel class of binding mols., which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (av. 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma exts. We have detd. the crystal structure of this DARPin at 1.7 Å, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An anal. of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.
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    Liu, S., Netzel-Arnett, S., Birkedal-Hansen, H., and Leppla, S. H. (2000) Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin. Cancer Res. 60, 6061– 6067
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    Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin
    Liu, Shihui; Netzel-Arnett, Sarah; Birkedal-Hansen, Henning; Leppla, Stephen H.
    Cancer Research (2000), 60 (21), 6061-6067CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed two mutated anthrax toxin protective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by MMPs. These MMP-targeted PA proteins were activated rapidly and selectively on the surface of MMP-overexpressing tumor cells. The activated PA proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A. The toxicity of the mutated PA proteins for MMP-overexpressing cells was blocked by hydroxamate inhibitors of MMPs, including BB94, and by a tissue inhibitor of matrix metalloproteinases (TIMP-2). The mutated PA proteins killed MMP-overexpressing tumor cells while sparing nontumorigenic normal cells when these were grown together in a coculture model, indicating that PA activation occurred on the tumor cell surface and not in the supernatant. This method of achieving cell-type specificity is conceptually distinct from, and potentially synergistic with, the more common strategy of retargeting a protein toxin by fusion to a growth factor, cytokine, or antibody.
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    Franke, D., Petoukhov, M. V., Konarev, P. V., Panjkovich, A., Tuukkanen, A., Mertens, H. D. T., Kikhney, A. G., Hajizadeh, N. R., Franklin, J. M., Jeffries, C. M., and Svergun, D. I. (2017) ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212– 1225,  DOI: 10.1107/S1600576717007786
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    ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions
    Franke, D.; Petoukhov, M. V.; Konarev, P. V.; Panjkovich, A.; Tuukkanen, A.; Mertens, H. D. T.; Kikhney, A. G.; Hajizadeh, N. R.; Franklin, J. M.; Jeffries, C. M.; Svergun, D. I.
    Journal of Applied Crystallography (2017), 50 (4), 1212-1225CODEN: JACGAR; ISSN:1600-5767. (International Union of Crystallography)
    A review. ATSAS is a comprehensive software suite for the anal. of small-angle scattering data from dil. solns. of biol. macromols. or nanoparticles. It contains applications for primary data processing and assessment, ab initio bead modeling, and model validation, as well as methods for the anal. of flexibility and mixts. In addn., approaches are supported that utilize information from X-ray crystallog., NMR spectroscopy or atomistic homol. modeling to construct hybrid models based on the scattering data. This article summarizes the progress made during the 2.5-2.8 ATSAS release series and highlights the latest developments. These include AMBIMETER, an assessment of the reconstruction ambiguity of exptl. data; DATCLASS, a multiclass shape classification based on exptl. data; SASRES, for estg. the resoln. of ab initio model reconstructions; CHROMIXS, a convenient interface to analyze in-line size exclusion chromatog. data; SHANUM, to evaluate the useful angular range in measured data; SREFLEX, to refine available high-resoln. models using normal mode anal.; SUPALM for a rapid superposition of low- and high-resoln. models; and SASPy, the ATSAS plugin for interactive modeling in PyMOL. All these features and other improvements are included in the ATSAS release 2.8, freely available for academic users from https://www.embl-hamburg.de/biosaxs/software.html.</a></a></a></a></a></a></a></a></a>.
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    Franke, D. and Svergun, D. I. (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342– 346,  DOI: 10.1107/S0021889809000338
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    DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering
    Franke, Daniel; Svergun, Dmitri I.
    Journal of Applied Crystallography (2009), 42 (2), 342-346CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
    DAMMIF, a revised implementation of the ab-initio shape-detn. program DAMMIN for small-angle scattering data, is presented. The program was fully rewritten, and its algorithm was optimized for speed of execution and modified to avoid limitations due to the finite search vol. Symmetry and anisometry constraints can be imposed on the particle shape, similar to DAMMIN. In equivalent conditions, DAMMIF is 25-40 times faster than DAMMIN on a single CPU. The possibility to utilize multiple CPUs is added to DAMMIF. The application is available in binary form for major platforms.
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    Volkov, V. V. and Svergun, D. I. (2003) Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860– 864,  DOI: 10.1107/S0021889803000268
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    Uniqueness of ab initio shape determination in small-angle scattering
    Volkov, Vladimir V.; Svergun, Dmitri I.
    Journal of Applied Crystallography (2003), 36 (3, Pt. 1), 860-864CODEN: JACGAR; ISSN:0021-8898. (Blackwell Munksgaard)
    Scattering patterns from geometrical bodies with different shapes and anisometry (solid and hollow spheres, cylinders, prisms) are computed and the shapes are reconstructed ab initio using envelope function and bead modeling methods. A procedure is described to analyze multiple solns. provided by bead modeling methods and to est. stability and reliability of the shape reconstruction. It is demonstrated that flat shapes are more difficult to restore than elongated ones and types of shapes are indicated, which require addnl. information for reliable shape reconstruction from the scattering data.
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    Webb, B., and Sali, A. (2017) Protein Structure Modeling with MODELLER, in Functional Genomics: Methods and Protocols, Methods in Molecular Biology, (Kaufmann, M., Klinger, C., and Savelsbergh, A., Eds.), pp 39– 54, Humana Press, New York, NY.
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    Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., De Beer, T. A. P., Rempfer, C., Bordoli, L., Lepore, R., and Schwede, T. (2018) SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296– W303,  DOI: 10.1093/nar/gky427
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    SWISS-MODEL: homology modelling of protein structures and complexes
    Waterhouse, Andrew; Bertoni, Martino; Bienert, Stefan; Studer, Gabriel; Tauriello, Gerardo; Gumienny, Rafal; Heer, Florian T.; de Beer, Tjaart A. P.; Rempfer, Christine; Bordoli, Lorenza; Lepore, Rosalba; Schwede, Torsten
    Nucleic Acids Research (2018), 46 (W1), W296-W303CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Homol. modeling has matured into an important technique in structural biol., significantly contributing to narrowing the gap between known protein sequences and exptl. detd. structures. Fully automated workflows and servers simplify and streamline the homol. modeling process, also allowing users without a specific computational expertise to generate reliable protein models and have easy access to modeling results, their visualization and interpretation. Here, we present an update to the SWISS-MODEL server, which pioneered the field of automated modeling 25 years ago and been continuously further developed. Recently, its functionality has been extended to the modeling of homo- and heteromeric complexes. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex are inferred by homol. modeling. Other major improvements include the implementation of a new modeling engine, ProMod3 and the introduction a new local model quality estn. method, QMEANDisCo.
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    Schweizer, A., Roschitzki-Voser, H., Amstutz, P., Briand, C., Gulotti-Georgieva, M., Prenosil, E., Binz, H. K., Capitani, G., Baici, A., Plückthun, A., and Grütter, M. G. (2007) Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. Structure 15, 625– 36,  DOI: 10.1016/j.str.2007.03.014
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    Inhibition of Caspase-2 by a Designed Ankyrin Repeat Protein: Specificity, Structure, and Inhibition Mechanism
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  • Abstract

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    Figure 1

    Figure 1. Properties and permeabilizing activity of Y406A. (a) A ribbon model of LLO 3D structure (PDB ID: 4CDB) with each domain (D1–D4) labeled in different color. Position of residue 406 is denoted by an orange color and an arrow. (b) A model of pore formation by Y406A (monomer binding to the membrane, oligomerization, pore formation). Domains are colored as on panel a. (c) Calcein release from large unilamellar vesicles after 30 min is shown at different pH values for LLO (black) and Y406A (red). Data reproduced with permission from Kisovec et al. (40) (d) Relative rate of hemolysis by 2.3 nM CDCs at pH 5.7 in the presence of DARPins: 5 μM D22 (red bars), 5 μM D22M (blue bar), a variant that was used for immobilization to the lipid membrane (see below), and 5 μM D6 and D30 (green bars), DARPin clones that were selected with ribosome display as high affinity binders, but did not inhibit hemolytic activity of Y406A. Mean ± SD; n = 2–7.

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    Figure 2

    Figure 2. Binding of D22 to Y406A in solution. (a) Size exclusion chromatogram of LLO and Y406A in the absence or presence of D22. Triangles indicate positions of elution peaks for different proteins. Note that LLO travels aberrantly on the size exclusion column eluting with larger volumes of elution buffer than expected. (b) Binding of D22 to LLO (gray) or Y406A (black) in solution (22 mM MES, 150 mM NaCl and 5 mM 2-mercaptoethanol, pH 5.7), measured by isothermal titration calorimetry. Top panel represents raw data of injections of 54.9 μM D22 into a 5.9 μM solution of LLO or Y406A. Bottom panel shows normalized integrated enthalpies plotted against the molar ratio. Circles represent experimental points, and the solid line corresponds to the best fit obtained by one-site reaction model. (c) An overlay of experimental scattering data obtained by SAXS experiment of Y406A-D22 complex (red circles) with the calculated scattering curve from the representative DAMMIF model (χ2 = 1.125, black line). Inset, overlay of Y406A (blue ribbon) and D22 (red ribbon) refined by rigid body modeling (χ2 = 1.12), with the best SAXS bead model in surface representation and the average SAXS bead model in mesh representation.

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    Figure 3

    Figure 3. Interaction of D22 with Y406A in the lipid membrane environment. (a) SPR measurements showing binding of 100 nM Y406A, 5 μM D22 and preincubated Y406A-D22 complex (with same concentrations of individual proteins as used for single proteins injections) to large unilamellar vesicles. (b) SPR sensorgrams of 5 μM D22 binding to 100 nM membrane-inserted Y406A or LLO. Membrane denotes control experiment with D22 binding to vesicles only. (c) Vesicle sedimentation assays with multilamellar vesicles after preincubation of LLO and D22, or Y406A and D22 in solution (“preincubated”) or when LLO or Y406A were first preincubated with vesicles (“membrane bound”). p, pellet; s, supernatant; w, additional washing step, which was included when assaying membrane bound LLO or Y406A in order to check for the completeness of binding. Band at app. 60 kDa corresponds to LLO or Y406A, while band at ca. 18 kDa corresponds to D22. D22 is present at 5× molar excess; therefore, a large portion of it is always unbound in supernatant. (d) Quantification of the SDS-PAGE data from (c) by densitometry. Full binding of D22 to Y406A (100%) was considered when one-fifth of the applied D22 was bound to Y406A. Mean ± SD; two sample t test, ***P < 0.001 (n = 4–6). Amount of bound D22 is reported when preincubated with LLO (1) or Y406A (2) in solution or when LLO (3) or Y406A (4) were first bound to vesicles.

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    Figure 4

    Figure 4. Modulation of Y406A permeabilization activity by D22 and pH. (a) Permeabilization of GUVs for FD10 at different conditions and induced by the 50 nM LLO. (b) Permeabilization induced by 50 nM Y406A. The graphs on the right in A and B show quantification of GUVs data from confocal microscopy images as represented on the left. Mean ± SD; n = 96–568. (c) Hemolysis induced by 18.2 nM LLO at different conditions. (d) Hemolysis induced by 18.2 nM Y406A. In C and D, mean ± SD is presented; n = 3. (e) 5 μM D22 by itself does not induce permeabilization of GUVs. Confocal images of GUVs on the left, and quantification is presented on the right. n is 256 and 458 for pH 6.5 and 8.0, respectively. (f) The truth table and schematic representation of a NOR logic gate for the Y406A-D22 system.

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    Figure 5

    Figure 5. Modulation of Y406A activity in calcein release experiments. (a,b) Calcein release from SUVs composed of POPC:Chol, 3:2 (mol:mol) as a result of pore formation by 1 μM LLO (a) or Y406A (b), in presence or absence of 5 μM D22 and at pH values 6.5 and 8.0. (c) Calcein release from LUVs composed of POPC:Chol, 1:1 (mol:mol) monitored at different conditions. Vesicles were stirred in 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 8.0. Final concentration of 30 nM Y406A (black triangle), 1 μM D22 or 3 μL of 7% HCl (to reduce pH to approximately 6.5) were added, respectively, at times denoted by triangles. The scale bar is the same for all fluorescent traces.

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    Figure 6

    Figure 6. Conjugation of D22M to vesicles and cleavage with different agents. Schematic diagrams of used systems and structures of employed lipids are shown. Y406A is presented with green color, D22 is shown in orange. Different parts of lipids used for conjugation are presented with different colors in the structural formulas and on the diagram. (a–c) A system employing DSPE-PEG2000Mal lipid, which attaches D22 to the lipid membrane and allows subsequent cleavage by MMP-9. (d–f) A system employing DSPE-PEG2000PDP lipid, which allows cleavage with reductant TCEP. The input of different proteins and reagents is shown above the sedimentation assay gels. The approximate positions of different proteins and lipids on the gels are indicated by arrows. i, 2 μg of D22M as an input; p and s denote pellet and supernatant after centrifugation of MLVs, respectively. (c) and (f) each represent one experiment where MLVs were first incubated with Y406A and then with MMP-9 or TCEP. 1, pellet after both incubations; 2, supernatant after centrifugation of MLVs after incubation with Y406A; 3, supernatant after centrifugation of MLVs with bound Y406A and incubation of MMP-9 or TCEP.

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    Figure 7

    Figure 7. A tunable vesicle system. (a) Y406A activity is controlled by pH and the reversible inhibitor D22. Vesicles are at pH 8.0; therefore, two input signals are needed for system activation: lowering pH with HCl and elimination of D22 from the system by release from vesicles induced by MMP-9 or TCEP. (b) A system based on DSPE-PEG2000Mal lipid. Bars represent fluorescence increase due to release of calcein from MLVs. A logic truth table and schematic representation of an AND gate is presented below the graph. (c) A system based on DSPE-PEG2000PDP lipid, which gives more flexibility for the removal of D22 from the vesicle membrane. A logic truth table and schematic representation of an OR-AND gate is presented beside the graph. n = 3–6, mean ± SD *P < 0.05; **P < 0.01.

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      Faulhammer, Dirk; Cukras, Anthony R.; Lipton, Richard J.; Landweber, Laura F.
      Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (4), 1385-1389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We have expanded the field of "DNA computers" to RNA and present a general approach for the soln. of satisfiability problems. As an example, we consider a variant of the "Knight problem," which asks generally what configurations of knights can one place on an n × n chess board such that no knight is attacking any other knight on the board. Using specific RNase digestion to manipulate strands of a 10-bit binary RNA library, we developed a mol. algorithm and applied it to a 3×3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine "bits" or placeholders in a combinatorial RNA library. We recovered a set of "winning" mols. that describe solns. to this problem.
    10. 10
      Choi, J. H. and Ostermeier, M. (2015) Rational design of a fusion protein to exhibit disulfide-mediated logic gate behavior. ACS Synth. Biol. 4, 400– 406,  DOI: 10.1021/sb500254g
      10
      Rational Design of a Fusion Protein to Exhibit Disulfide-Mediated Logic Gate Behavior
      Choi, Jay H.; Ostermeier, Marc
      ACS Synthetic Biology (2015), 4 (4), 400-406CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Synthetic cellular logic gates are primarily built from gene circuits owing to their inherent modularity. Single proteins can also possess logic gate functions and offer the potential to be simpler, quicker, and less dependent on cellular resources than gene circuits. However, the design of protein logic gates that are modular and integrate with other cellular components is a considerable challenge. As a step toward addressing this challenge, we describe the design, construction, and characterization of AND, ORN, and YES logic gates built by introducing disulfide bonds into RG13, a fusion of maltose binding protein and TEM-1 β-lactamase for which maltose is an allosteric activator of enzyme activity. We rationally designed these disulfide bonds to manipulate RG13's allosteric regulation mechanism such that the gating had maltose and reducing agents as input signals, and the gates could be toggled between different gating functions using redox agents, although some gates performed suboptimally.
    11. 11
      Fink, T., Lonzarić, J., Praznik, A., Plaper, T., Merljak, E., Leben, K., Jerala, N., Lebar, T., Strmšek, Ž., Lapenta, F., Benčina, M., and Jerala, R. (2019) Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat. Chem. Biol. 15, 115– 122,  DOI: 10.1038/s41589-018-0181-6
      11
      Design of fast proteolysis-based signaling and logic circuits in mammalian cells
      Fink, Tina; Lonzaric, Jan; Praznik, Arne; Plaper, Tjasa; Merljak, Estera; Leben, Katja; Jerala, Nina; Lebar, Tina; Strmsek, Ziga; Lapenta, Fabio; Bencina, Mojca; Jerala, Roman
      Nature Chemical Biology (2019), 15 (2), 115-122CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, the authors present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chem. or biol. signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.
    12. 12
      Moseley, F., Halámek, J., Kramer, F., Poghossian, A., Schöning, M. J., and Katz, E. (2014) An enzyme-based reversible CNOT logic gate realized in a flow system. Analyst 139, 1839– 1842,  DOI: 10.1039/c4an00133h
      12
      An enzyme-based reversible CNOT logic gate realized in a flow system
      Moseley, Fiona; Halamek, Jan; Kramer, Friederike; Poghossian, Arshak; Schoening, Michael J.; Katz, Evgeny
      Analyst (Cambridge, United Kingdom) (2014), 139 (8), 1839-1842CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD+ and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomol. realization of a CNOT gate is promising for integration into complex biomol. networks and future biosensor/biomedical applications.
    13. 13
      Stein, V. and Alexandrov, K. (2014) Protease-based synthetic sensing and signal amplification. Proc. Natl. Acad. Sci. U. S. A. 111, 15934– 15939,  DOI: 10.1073/pnas.1405220111
      13
      Protease-based synthetic sensing and signal amplification
      Stein, Viktor; Alexandrov, Kirill
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (45), 15934-15939CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The bottom-up design of protein-based signaling networks is a key goal of synthetic biol.; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based mol. switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously obsd. in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify mol. queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biol. process.
    14. 14
      Zhou, J., Arugula, M. A., Halámek, J., Pita, M., and Katz, E. (2009) Enzyme-based NAND and NOR logic gates with modular design. J. Phys. Chem. B 113, 16065– 16070,  DOI: 10.1021/jp9079052
      14
      Enzyme-Based NAND and NOR Logic Gates with Modular Design
      Zhou, Jian; Arugula, Mary A.; Halamek, Jan; Pita, Marcos; Katz, Evgeny
      Journal of Physical Chemistry B (2009), 113 (49), 16065-16070CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      The logic gates NAND/NOR were mimicked by enzyme biocatalyzed reactions activated by sucrose, maltose and phosphate. The subunits performing AND/OR Boolean logic operations were designed using maltose phosphorylase and cooperative work of invertase/amyloglucosidase, resp. Glucose produced as the output signal from the AND/OR subunits was applied as the input signal for the INVERTER gate composed of alc. dehydrogenase, glucose oxidase, microperoxidase-11, ethanol and NAD+, which generated the final output in the form of NADH inverting the logic signal from 0 to 1 or from 1 to 0. The final output signal was amplified by a self-promoting biocatalytic system. In order to fulfill the Boolean properties of associativity and commutativity in logic networks, the final NADH output signal was converted to the initial signals of maltose and phosphate, thus allowing assembling of the same std. units in concatenated sequences. The designed modular approach, signal amplification and conversion processes open the way toward complex logic networks composed of std. elements resembling electronic integrated circuitries.
    15. 15
      Gao, X. J., Chong, L. S., Kim, M. S., and Elowitz, M. B. (2018) Programmable protein circuits in living cells. Science 361, 1252– 1258,  DOI: 10.1126/science.aat5062
      15
      Programmable protein circuits in living cells
      Gao, Xiaojing J.; Chong, Lucy S.; Kim, Matthew S.; Elowitz, Michael B.
      Science (Washington, DC, United States) (2018), 361 (6408), 1252-1258CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnol. applications.
    16. 16
      Hilburger, C. E., Jacobs, M. L., Lewis, K. R., Peruzzi, J. A., and Kamat, N. P. (2019) Controlling Secretion in Artificial Cells with a Membrane and Gate. ACS Synth. Biol. 8, 1224– 1230,  DOI: 10.1021/acssynbio.8b00435
      16
      Controlling Secretion in Artificial Cells with a Membrane AND Gate
      Hilburger, Claire E.; Jacobs, Miranda L.; Lewis, Kamryn R.; Peruzzi, Justin A.; Kamat, Neha P.
      ACS Synthetic Biology (2019), 8 (6), 1224-1230CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The assembly of channel proteins into vesicle membranes is a useful strategy to control activities of vesicle-based systems. Here, the authors developed a membrane AND gate that responds to both a fatty acid and a pore-forming channel protein to induce the release of encapsulated cargo. The authors explored how membrane compn. affects the functional assembly of α-hemolysin into phospholipid vesicles as a function of oleic acid content and α-hemolysin concn. The authors then showed that the authors could induce α-hemolysin assembly when the authors added oleic acid micelles to a specific compn. of phospholipid vesicles. Finally, the authors demonstrated that the membrane AND gate could be coupled to a gene expression system. The study provides a new method to control the temporal dynamics of vesicle permeability by controlling when the functional assembly of a channel protein into synthetic vesicles occurs. Furthermore, a membrane AND gate that utilizes membrane-assocg. biomols. introduces a new way to implement Boolean logic that should complement genetic logic circuits and ultimately enhance the capabilities of artificial cellular systems.
    17. 17
      Adamala, K. P., Martin-Alarcon, D. A., Guthrie-Honea, K. R., and Boyden, E. S. (2017) Engineering genetic circuit interactions within and between synthetic minimal cells. Nat. Chem. 9, 431– 439,  DOI: 10.1038/nchem.2644
      17
      Engineering genetic circuit interactions within and between synthetic minimal cells
      Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
      Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
    18. 18
      Einfalt, T., Goers, R., Dinu, I. A., Najer, A., Spulber, M., Onaca-Fischer, O., and Palivan, C. G. (2015) Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes. Nano Lett. 15, 7596– 7603,  DOI: 10.1021/acs.nanolett.5b03386
      18
      Stimuli-Triggered Activity of Nanoreactors by Biomimetic Engineering Polymer Membranes
      Einfalt, Tomaz; Goers, Roland; Dinu, Ionel Adrian; Najer, Adrian; Spulber, Mariana; Onaca-Fischer, Ozana; Palivan, Cornelia G.
      Nano Letters (2015), 15 (11), 7596-7603CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The development of advanced stimuli-responsive systems for medicine, catalysis, or technol. requires compartmentalized reaction spaces with triggered activity. Only very few stimuli-responsive systems preserve the compartment architecture, and none allows a triggered activity in situ. We present here a biomimetic strategy to mol. transmembrane transport by engineering synthetic membranes equipped with channel proteins so that they are stimuli-responsive. Nanoreactors with triggered activity were designed by simultaneously encapsulating an enzyme inside polymer compartments, and inserting protein "gates" in the membrane. The outer membrane protein F (OmpF) porin was chem. modified with a pH-responsive mol. cap to serve as "gate" producing pH-driven mol. flow through the membrane and control the in situ enzymic activity. This strategy provides complex reaction spaces necessary in "smart" medicine and for biomimetic engineering of artificial cells.
    19. 19
      Einfalt, T., Witzigmann, D., Edlinger, C., Sieber, S., Goers, R., Najer, A., Spulber, M., Onaca-Fischer, O., Huwyler, J., and Palivan, C. G. (2018) Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment. Nat. Commun. 9, 1127,  DOI: 10.1038/s41467-018-03560-x
      19
      Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment
      Einfalt T; Edlinger C; Goers R; Najer A; Spulber M; Onaca-Fischer O; Palivan C G; Einfalt T; Witzigmann D; Sieber S; Huwyler J; Goers R
      Nature communications (2018), 9 (1), 1127 ISSN:.
      Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.
    20. 20
      van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008) Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112– 124,  DOI: 10.1038/nrm2330
      20
      Membrane lipids: where they are and how they behave
      van Meer, Gerrit; Voelker, Dennis R.; Feigenson, Gerald W.
      Nature Reviews Molecular Cell Biology (2008), 9 (2), 112-124CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Throughout the biol. world, a 30 Å hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochem. and biophys. findings have provided a detailed model of the compn. and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases, and transporters, plus the intrinsic phys. phase behavior of lipids and their interactions with membrane proteins, to create the unique compns. and multiple functionalities of their individual membranes.
    21. 21
      Golynskiy, M. V., Koay, M. S., Vinkenborg, J. L., and Merkx, M. (2011) Engineering protein switches: sensors, regulators, and spare parts for biology and biotechnology. ChemBioChem 12, 353– 361,  DOI: 10.1002/cbic.201000642
      21
      Engineering Protein Switches: Sensors, Regulators, and Spare Parts for Biology and Biotechnology
      Golynskiy, Misha V.; Koay, Melissa S.; Vinkenborg, Jan L.; Merkx, Maarten
      ChemBioChem (2011), 12 (3), 353-361CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The authors review the underlying engineering concepts for constructing protein switches, most of which are inspired by or reminiscent of natural examples. The authors focus on those strategies that have the potential to be generally applicable, and discuss them according to their mechanism of action in order of increasing modularity and decreasing domain integration. Finally, the authors identify the opportunities and challenges assocd. with these different engineering concepts and discuss future perspectives.
    22. 22
      Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L., and Beltram, F. (2010) Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat. Methods 7, 516– 518,  DOI: 10.1038/nmeth.1471
      22
      Simultaneous intracellular chloride and pH measurements using a GFP-based sensor
      Arosio, Daniele; Ricci, Fernanda; Marchetti, Laura; Gualdani, Roberta; Albertazzi, Lorenzo; Beltram, Fabio
      Nature Methods (2010), 7 (7), 516-518CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concn. in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.
    23. 23
      Dal Peraro, M. and van der Goot, F. G. (2016) Pore-forming toxins: ancient, but never really out of fashion. Nat. Rev. Microbiol. 14, 77– 92,  DOI: 10.1038/nrmicro.2015.3
      23
      Pore-forming toxins: ancient, but never really out of fashion
      Dal Peraro Matteo; van der Goot F Gisou
      Nature reviews. Microbiology (2016), 14 (2), 77-92 ISSN:.
      Pore-forming toxins (PFTs) are virulence factors produced by many pathogenic bacteria and have long fascinated structural biologists, microbiologists and immunologists. Interestingly, pore-forming proteins with remarkably similar structures to PFTs are found in vertebrates and constitute part of their immune system. Recently, structural studies of several PFTs have provided important mechanistic insights into the metamorphosis of PFTs from soluble inactive monomers to cytolytic transmembrane assemblies. In this Review, we discuss the diverse pore architectures and membrane insertion mechanisms that have been revealed by these studies, and we consider how these features contribute to binding specificity for different membrane targets. Finally, we explore the potential of these structural insights to enable the development of novel therapeutic strategies that would prevent both the establishment of bacterial resistance and an excessive immune response.
    24. 24
      Anderluh, G. and Lakey, J. H. (2008) Disparate proteins use similar architectures to damage membranes. Trends Biochem. Sci. 33, 482– 490,  DOI: 10.1016/j.tibs.2008.07.004
      24
      Disparate proteins use similar architectures to damage membranes
      Anderluh, Gregor; Lakey, Jeremy H.
      Trends in Biochemical Sciences (2008), 33 (10), 482-490CODEN: TBSCDB; ISSN:0968-0004. (Elsevier B.V.)
      A review. Membrane disruption can efficiently alter cellular function; indeed, pore-forming toxins (PFTs) are well known as important bacterial virulence factors. However, recent data have revealed that structures similar to those found in PFTs are found in membrane active proteins across disparate phyla. Many similarities can be identified only at the 3D-structural level. Of note, domains found in membrane-attack complex proteins of complement and perforin (MACPF) resemble cholesterol-dependent cytolysins from Gram-pos. bacteria, and the Bcl family of apoptosis regulators share similar architectures with Escherichia coli pore-forming colicins. These and other correlations provide considerable help in understanding the structural requirements for membrane binding and pore formation.
    25. 25
      Laventie, B., Potrich, C., Atmanène, C., Saleh, M., Joubert, O., Viero, G., Bachmeyer, C., Antonini, V., Mancini, I., Cianferani-Sanglier, S., Keller, D., Colin, D. A., Bourcier, T., Anderluh, G., van Dorsselaer, A., Dalla Serra, M., and Prévost, G. (2013) p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions. Biochem. J. 450, 559– 571,  DOI: 10.1042/BJ20121628
      25
      p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions
      Laventie, Benoit-Joseph; Potrich, Cristina; Atmanene, Cedric; Saleh, Maher; Joubert, Olivier; Viero, Gabriella; Bachmeyer, Christoph; Antonini, Valeria; Mancini, Ines; Cianferani-Sanglier, Sarah; Keller, Daniel; Colin, Didier A.; Bourcier, Tristan; Anderluh, Gregor; van Dorsselaer, Alain; Dalla Serra, Mauro; Prevost, Gilles
      Biochemical Journal (2013), 450 (3), 559-571CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus β-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chems. have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 μM for Ca2+ activation and of 2-50 μM for cell lysis. SCns were obsd. to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramol. interactions (1:1 stoichiometry) between SCns and class S proteins in soln., but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concns. according to the no. of benzenes (30-300 μM; SC8>SC6»SC4). The inhibitory properties of SCns were also obsd. in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.
    26. 26
      Booth, M. J., Schild, V. R., Graham, A. D., Olof, S. N., and Bayley, H. (2016) Light-activated communication in synthetic tissues. Sci. Adv. 2, e1600056  DOI: 10.1126/sciadv.1600056
      26
      Light-activated communication in synthetic tissues
      Booth, Michael J.; Schild, Vanessa Restrepo; Graham, Alexander D.; Olof, Sam N.; Bayley, Hagan
      Science Advances (2016), 2 (4), e1600056/1-e1600056/11CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)
      We have previously used three-dimensional (3D) printing to prep. tissue-like materials in which picoliter aq. compartments are sepd. by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional elec. communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way.
    27. 27
      Provoda, C. J., Stier, E. M., and Lee, K. D. (2003) Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin. J. Biol. Chem. 278, 35102– 35108,  DOI: 10.1074/jbc.M305411200
      27
      Tumor cell killing enabled by Listeriolysin O-liposome-mediated delivery of the protein toxin gelonin
      Provoda, Chester J.; Stier, Ethan M.; Lee, Kyung-Dall
      Journal of Biological Chemistry (2003), 278 (37), 35102-35108CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromol., however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degrdn. within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro expts., co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of ∼0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equiv. concns. of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into s.c. solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes contg. both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.
    28. 28
      Robertson, J. W. F., Rodrigues, C. G., Stanford, V. M., Rubinson, K. A., Krasilnikov, O. V., and Kasianowicz, J. J. (2007) Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl. Acad. Sci. U. S. A. 104, 8207– 8211,  DOI: 10.1073/pnas.0611085104
      28
      Single-molecule mass spectrometry in solution using a solitary nanopore
      Robertson, Joseph W. F.; Rodrigues, Claudio G.; Stanford, Vincent M.; Rubinson, Kenneth A.; Krasilnikov, Oleg V.; Kasianowicz, John J.
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (20), 8207-8211CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The authors introduce a 2-dimensional method for mass spectrometry in soln. that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) mols. that enter a single α-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of mols. in soln.
    29. 29
      Clarke, J., Wu, H., Jayasinghe, L., Patel, A., Reid, S., and Bayley, H. (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265– 270,  DOI: 10.1038/nnano.2009.12
      29
      Continuous base identification for single-molecule nanopore DNA sequencing
      Clarke, James; Wu, Hai-Chen; Jayasinghe, Lakmal; Patel, Alpesh; Reid, Stuart; Bayley, Hagan
      Nature Nanotechnology (2009), 4 (4), 265-270CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      A single-mol. method for sequencing DNA that does not require fluorescent labeling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide mols. from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, the authors show that a protein nanopore with a covalently attached adapter mol. can continuously identify unlabeled nucleoside 5'-monophosphate mols. with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four std. DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analyzing epigenetic modifications.
    30. 30
      Wang, Y., Montana, V., Grubišić, V., Stout, R. F., Parpura, V., and Gu, L. Q. (2015) Nanopore sensing of botulinum toxin type B by discriminating an enzymatically cleaved peptide from a synaptic protein synaptobrevin 2 derivative. ACS Appl. Mater. Interfaces 7, 184– 192,  DOI: 10.1021/am5056596
      30
      Nanopore Sensing of Botulinum Toxin Type B by Discriminating an Enzymatically Cleaved Peptide from a Synaptic Protein Synaptobrevin 2 Derivative
      Wang, Yong; Montana, Vedrana; Grubisic, Vladimir; Stout, Randy F.; Parpura, Vladimir; Gu, Li-Qun
      ACS Applied Materials & Interfaces (2015), 7 (1), 184-192CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and assocd. with legal and ethical constrains. Various biochem., optical and mech. methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin's enzymic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 deriv. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concn. was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophys. mechanisms for biopolymer transportation.
    31. 31
      Wang, Y., Zheng, D., Tan, Q., Wang, M., and Gu, L. (2011) Nanopore-based detection of circulating microRNAs in lung cancer patients. Nat. Nanotechnol. 6, 668– 674,  DOI: 10.1038/nnano.2011.147
      31
      Nanopore-based detection of circulating microRNAs in lung cancer patients
      Wang, Yong; Zheng, Da-Li; Tan, Qiu-Lin; Wang, Michael X.; Gu, Li-Qun
      Nature Nanotechnology (2011), 6 (10), 668-674CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      MicroRNAs are short RNA mols. that regulate gene expression, and have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, detecting microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the α-haemolysin protein can selectively detect microRNAs at the single mol. level in plasma samples from lung cancer patients without the need for labels or amplification of the microRNA. The sensor, which uses a programmable oligonucleotide probe to generate a target-specific signature signal, can quantify subpicomolar levels of cancer-assocd. microRNAs and can distinguish single-nucleotide differences between microRNA family members. This approach is potentially useful for quant. microRNA detection, the discovery of disease markers and non-invasive early diagnosis of cancer.
    32. 32
      Braha, O., Bayley, H., Gu, L. Q., Zhou, L., Lu, X., and Cheley, S. (2000) Simultaneous stochastic sensing of divalent metal ions. Nat. Biotechnol. 18, 1005– 1007,  DOI: 10.1038/79275
      32
      Simultaneous stochastic sensing of divalent metal ions
      Braha, Orit; Gu, Lin-Qun; Zhou, Li; Lu, Xiaofeng; Cheley, Stephen; Bayley, Hagan
      Nature Biotechnology (2000), 18 (9), 1005-1007CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      Stochastic sensing is an emerging anal. technique that relies upon single-mol. detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concn. of an analyte and, through a characteristic signature, its identity. Here, we show that the concns. of two or more divalent metal ions in soln. can be detd. simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.
    33. 33
      Villar, G., Graham, A. D., and Bayley, H. (2013) A tissue-like printed material. Science 340, 48– 52,  DOI: 10.1126/science.1229495
      33
      A Tissue-Like Printed Material
      Villar, Gabriel; Graham, Alexander D.; Bayley, Hagan
      Science (Washington, DC, United States) (2013), 340 (6128), 48-52CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Living cells communicate and cooperate to produce the emergent properties of tissues. Synthetic mimics of cells, such as liposomes, are typically incapable of cooperation and therefore cannot readily display sophisticated collective behavior. We printed tens of thousands of picoliter aq. droplets that become joined by single lipid bilayers to form a cohesive material with cooperating compartments. Three-dimensional structures can be built with heterologous droplets in software-defined arrangements. The droplet networks can be functionalized with membrane proteins; for example, to allow rapid elec. communication along a specific path. The networks can also be programmed by osmolarity gradients to fold into otherwise unattainable designed structures. Printed droplet networks might be interfaced with tissues, used as tissue engineering substrates, or developed as mimics of living tissue.
    34. 34
      Elani, Y., Law, R. V., and Ces, O. (2014) Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 5, 5305,  DOI: 10.1038/ncomms6305
      34
      Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways
      Elani, Yuval; Law, Robert V.; Ces, Oscar
      Nature Communications (2014), 5 (), 5305CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      In the discipline of bottom-up synthetic biol., vesicles define the boundaries of artificial cells and are increasingly being used as biochem. microreactors operating in physiol. environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chem. pathway to be sepd. in space, this platform bridges the gap between table-top chem. and chem. that is performed within vesicles.
    35. 35
      Hamon, M. A., Ribet, D., Stavru, F., and Cossart, P. (2012) Listeriolysin O: the Swiss army knife of Listeria. Trends Microbiol. 20, 360– 368,  DOI: 10.1016/j.tim.2012.04.006
      35
      Listeriolysin O: the Swiss army knife of Listeria
      Hamon, Melanie Anne; Ribet, David; Stavru, Fabrizia; Cossart, Pascale
      Trends in Microbiology (2012), 20 (8), 360-368CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      A review. Listeriolysin O (LLO) is a toxin produced by Listeria monocytogenes, an opportunistic bacterial pathogen responsible for the disease listeriosis. This disease starts with the ingestion of contaminated foods and mainly affects immunocompromised individuals, newborns, and pregnant women. In the lab., L. monocytogenes is used as a model organism to study processes such as cell invasion, intracellular survival, and cell-to-cell spreading, as this Gram-pos. bacterium has evolved elaborate mol. strategies to subvert host cell functions. LLO is a major virulence factor originally shown to be crucial for bacterial escape from the internalization vacuole after entry into cells. However, recent studies are revisiting the role of LLO during infection and are revealing new insights into the action of LLO, in particular before bacterial entry. These latest findings along with their impact on the infectious process will be discussed.
    36. 36
      Köster, S., van Pee, K., Hudel, M., Leustik, M., Rhinow, D., Kühlbrandt, W., Chakraborty, T., and Yildiz, O. (2014) Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation. Nat. Commun. 5, 3690,  DOI: 10.1038/ncomms4690
      36
      Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation
      Koster Stefan; van Pee Katharina; Rhinow Daniel; Kuhlbrandt Werner; Yildiz Ozkan; Hudel Martina; Leustik Martin; Chakraborty Trinad
      Nature communications (2014), 5 (), 3690 ISSN:.
      Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca(2+) oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca(2+) uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.
    37. 37
      Ruan, Y., Rezelj, S., Bedina Zavec, A., Anderluh, G., and Scheuring, S. (2016) Listeriolysin O membrane damaging activity involves arc formation and lineaction - implication for Listeria monocytogenes escape from phagocytic vacuole. PLoS Pathog. 12, e1005597  DOI: 10.1371/journal.ppat.1005597
      37
      Listeriolysin O membrane damaging activity involves arc formation and lineaction - implication for Listeria monocytogenes escape from phagocytic vacuole
      Ruan, Yi; Rezelj, Sasa; Zavec, Apolonija Bedina; Anderluh, Gregor; Scheuring, Simon
      PLoS Pathogens (2016), 12 (4), e1005597/1-e1005597/18CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      Listeriolysin-O (LLO) plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood, probably due to unavailability of suitable exptl. tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed at. force microscopy (HS-AFM) featuring high spatio-temporal resoln. on model membranes and optical microscopy on giant unilamellar vesicles (GUVs) to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concn. and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context.
    38. 38
      Podobnik, M., Marchioretto, M., Zanetti, M., Bavdek, A., Kisovec, M., Cajnko, M. M., Lunelli, L., Serra, M. D., and Anderluh, G. (2015) Plasticity of lysteriolysin O pores and its regulation by pH and unique histidine. Sci. Rep. 5, 9623,  DOI: 10.1038/srep09623
      38
      Plasticity of Listeriolysin O Pores and its Regulation by pH and Unique Histidine
      Podobnik, Marjetka; Marchioretto, Marta; Zanetti, Manuela; Bavdek, Andrej; Kisovec, Matic; Cajnko, Misa Mojca; Lunelli, Lorenzo; Dalla Serra, Mauro; Anderluh, Gregor
      Scientific Reports (2015), 5 (), 9623/1-9623/10CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irresp. of pH. While pore diams. at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.
    39. 39
      Mulvihill, E., Van Pee, K., Mari, S. A., Müller, D. J., and Yildiz, Ö. (2015) Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O. Nano Lett. 15, 6965– 6973,  DOI: 10.1021/acs.nanolett.5b02963
      39
      Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O
      Mulvihill Estefania; Mari Stefania A; Muller Daniel J; van Pee Katharina; Yildiz Ozkan
      Nano letters (2015), 15 (10), 6965-73 ISSN:.
      Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.
    40. 40
      Kisovec, M., Rezelj, S., Knap, P., Cajnko, M., Caserman, S., Flašker, A., Žnidaršič, N., Repič, M., Mavri, J., Ruan, Y., Scheuring, S., Podobnik, M., and Anderluh, G. (2017) Engineering a pH responsive pore forming protein. Sci. Rep. 7, 42231,  DOI: 10.1038/srep42231
      40
      Engineering a pH responsive pore forming protein
      Kisovec, Matic; Rezelj, Sasa; Knap, Primoz; Cajnko, Misa Mojca; Caserman, Simon; Flasker, Ajda; Znidarsic, Nada; Repic, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor
      Scientific Reports (2017), 7 (), 42231CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural anal. coupled with mol. dynamics and mutational anal., we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Mol. dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnol. applications such as controlled release of substances or as a sensor of environmental pH.
    41. 41
      Dreier, B., and Plückthun, A. (2011) Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries, in PCR Protocols. Methods in Molecular Biology (Methods and Protocols), (Park, D. J., Ed.), pp 283– 306, Humana Press, Totowa, NJ.
      There is no corresponding record for this reference.
    42. 42
      Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A. (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575– 82,  DOI: 10.1038/nbt962
      42
      High-affinity binders selected from designed ankyrin repeat protein libraries
      Binz, H. Kaspar; Amstutz, Patrick; Kohl, Andreas; Stumpp, Michael T.; Briand, Christophe; Forrer, Patrik; Gruetter, Markus G.; Plueckthun, Andreas
      Nature Biotechnology (2004), 22 (5), 575-582CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat nos. with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and detd. the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 Å resoln. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding mols. and, because of the very favorable biophys. properties of the designed AR proteins, an attractive alternative to antibody libraries.
    43. 43
      Bavdek, A., Gekara, N. O., Priselac, D., Gutiérrez Aguirre, I., Darji, A., Chakraborty, T., Macek, P., Lakey, J. H., Weiss, S., and Anderluh, G. (2007) Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O. Biochemistry 46, 4425– 4437,  DOI: 10.1021/bi602497g
      43
      Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O
      Bavdek, Andrej; Gekara, Nelson O.; Priselac, Dragan; Gutierrez Aguirre, Ion; Darji, Ayub; Chakraborty, Trinad; Macek, Peter; Lakey, Jeremy H.; Weiss, Siegfried; Anderluh, Gregor
      Biochemistry (2007), 46 (14), 4425-4437CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concns. in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiol. or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approx. 35 mol % cholesterol, a concn. range typically found in lipid rafts. The narrow transition region of cholesterol concn. sepg. low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is detd. by the most exposed 3β-hydroxy group of cholesterol. In addn., LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiol. or slightly basic pH values. The results clearly show that LLO activity at physiol. pH cannot be neglected and that its action at sites distal to cell entry may have important physiol. consequences for Listeria pathogenesis.
    44. 44
      Sallee, N. A., Yeh, B. J., and Lim, W. A. (2007) Engineering modular protein interaction switches by sequence overlap. J. Am. Chem. Soc. 129, 4606– 4611,  DOI: 10.1021/ja0672728
      44
      Engineering Modular Protein Interaction Switches by Sequence Overlap
      Sallee, Nathan A.; Yeh, Brian J.; Lim, Wendell A.
      Journal of the American Chemical Society (2007), 129 (15), 4606-4611CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Many cellular signaling pathways contain proteins whose interactions change in response to upstream inputs, allowing for conditional activation or repression of the interaction based on the presence of the input mol. The ability to engineer similar regulation into protein interaction elements would provide us with powerful tools for controlling cell signaling. Here we describe an approach for engineering diverse synthetic protein interaction switches. Specifically, by overlapping the sequences of pairs of protein interaction domains and peptides, we have been able to generate mutually exclusive regulation over their interactions. Thus, the hybrid protein (which is composed of the two overlapped interaction modules) can bind to either of the two resp. ligands for those modules, but not to both simultaneously. We show that these synthetic switch proteins can be used to regulate specific protein-protein interactions in vivo. These switches allow us to disrupt an interaction with the addn. or activation of a protein input that has no natural connection to the interaction in question. Therefore, they give us the ability to make novel connections between normally unrelated signaling pathways and to rewire the input/output relationships of cellular behaviors. Our expts. also suggest a possible mechanism by which complex regulatory proteins might have evolved from simpler components.
    45. 45
      Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., and Hahn, K. M. (2010) Engineered allosteric activation of kinases in living cells. Nat. Biotechnol. 28, 743– 747,  DOI: 10.1038/nbt.1639
      45
      Engineered allosteric activation of kinases in living cells
      Karginov, Andrei V.; Ding, Feng; Kota, Pradeep; Dokholyan, Nikolay V.; Hahn, Klaus M.
      Nature Biotechnology (2010), 28 (7), 743-747CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions. Catalytic activity is restored by addn. of rapamycin or non-immunosuppresive rapamycin analogs. Mol. modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.
    46. 46
      Edwards, W. R., Busse, K., Allemann, R. K., and Jones, D. D. (2008) Linking the functions of unrelated proteins using a novel directed evolution domain insertion method. Nucleic Acids Res. 36, e78  DOI: 10.1093/nar/gkn363
      46
      Linking the functions of unrelated proteins using a novel directed evolution domain insertion method
      Edwards, Wayne R.; Busse, Kathy; Allemann, Rudolf K.; Jones, D. Dafydd
      Nucleic Acids Research (2008), 36 (13), e78/1-e78/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the sep. protein activities, thus providing a general strategy for constructing mol. switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were obsd. in loops, several also occurred close to the termini of α-helixes and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.
    47. 47
      Ha, J. H., Butler, J. S., Mitrea, D. M., and Loh, S. N. (2006) Modular enzyme design: Regulation by mutually exclusive protein folding. J. Mol. Biol. 357, 1058– 1062,  DOI: 10.1016/j.jmb.2006.01.073
      47
      Modular Enzyme Design: Regulation by Mutually Exclusive Protein Folding
      Ha, Jeung-Hoi; Butler, James S.; Mitrea, Diana M.; Loh, Stewart N.
      Journal of Molecular Biology (2006), 357 (4), 1058-1062CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
      A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topol. constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodn. tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.
    48. 48
      Tweten, R. K., Hotze, E. M., and Wade, K. R. (2015) The unique molecular choreography of giant pore formation by the cholesterol-dependent cytolysins of Gram-positive bacteria. Annu. Rev. Microbiol. 69, 323– 340,  DOI: 10.1146/annurev-micro-091014-104233
      48
      The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria
      Tweten, Rodney K.; Hotze, Eileen M.; Wade, Kristin R.
      Annual Review of Microbiology (2015), 69 (), 323-340CODEN: ARMIAZ; ISSN:0066-4227. (Annual Reviews)
      A review. The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophys., and biochem. analyses revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. The authors' knowledge of the mol. details of these dramatic structural changes in CDCs has transformed the understanding of how giant pore complexes are assembled and has been crit. to the understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs were employed in pore-independent processes.
    49. 49
      Song, L., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley, H., and Gouaux, J. E. (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859– 1866,  DOI: 10.1126/science.274.5294.1859
      49
      Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore
      Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric
      Science (Washington, D. C.) (1996), 274 (5294), 1859-1866CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The structure of the Staphylococcus aureus α-hemolysin pore has been detd. to 1.9 Å resoln. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 Å in length, that runs along the sevenfold axis and ranges from 14 Å to 46 Å in diam. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each promoter contributes two β strands, each 65 Å long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 Å wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-sol. protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.
    50. 50
      Podobnik, M., Savory, P., Rojko, N., Kisovec, M., Wood, N., Hambley, R., Pugh, J., Wallace, E. J., McNeill, L., Bruce, M., Liko, I., Allison, T. M., Mehmood, S., Yilmaz, N., Kobayashi, T., Gilbert, R. J. C., Robinson, C. V., Jayasinghe, L., and Anderluh, G. (2016) Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat. Commun. 7, 11598,  DOI: 10.1038/ncomms11598
      50
      Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly
      Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E. Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M.; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J. C.; Robinson, Carol V.; Jayasinghe, Lakmal; Anderluh, Gregor
      Nature Communications (2016), 7 (), 11598CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of addnl. luminal compartments as commonly found in other toxin pores. Mutagenic anal. and at. force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.
    51. 51
      Tsuchikama, K. and An, Z. (2018) Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell 9, 33– 46,  DOI: 10.1007/s13238-016-0323-0
      51
      Antibody-drug conjugates: recent advances in conjugation and linker chemistries
      Tsuchikama, Kyoji; An, Zhiqiang
      Protein & Cell (2018), 9 (1), 33-46CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press)
      The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small mols. (payloads) through chem. linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based mol. platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris and Kadcyla, this drug class has been rapidly growing along with about 60 ADCs currently in clin. trials. In this article, we briefly review mol. aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clin. effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into mol. design and strategies for clin. effective ADCs from medicinal chem. and pharmacol. standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an esp. promising path to improving ADC design, which will open the way for novel cancer therapeutics.
    52. 52
      Leriche, G., Chisholm, L., and Wagner, A. (2012) Cleavable linkers in chemical biology. Bioorg. Med. Chem. 20, 571– 582,  DOI: 10.1016/j.bmc.2011.07.048
      52
      Cleavable linkers in chemical biology
      Leriche, Geoffray; Chisholm, Louise; Wagner, Alain
      Bioorganic & Medicinal Chemistry (2012), 20 (2), 571-582CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      A review. Interest in cleavable linkers is growing due to the rapid development and expansion of chem. biol. The chem. constrains imposed by the biol. conditions cause significant challenges for org. chemists. In this review we will present an overview of the cleavable linkers used in chem. biol. classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradn., electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.
    53. 53
      Cajnko, M. M., Marušić, M., Kisovec, M., Rojko, N., Benčina, M., Caserman, S., and Anderluh, G. (2015) Listeriolysin O affects the permeability of Caco-2 monolayer in a pore-dependent and Ca2+-independent manner. PLoS One 10, e0130471  DOI: 10.1371/journal.pone.0130471
      53
      Listeriolysin O affects the permeability of Caco-2 monolayer in a pore-dependent and Ca2+-independent manner
      Cajnko Misa, Mojca; Marusic, Maja; Kisovec, Matic; Rojko, Nejc; Bencina, Mojca; Caserman, Simon; Anderluh, Gregor
      PLoS One (2015), 10 (6), e0130471/1-e0130471/21CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Apical application of both toxins causes a significant drop in transepithelial elec. resistance (TEER), with higher LLO concns. or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and assocd. actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was obsd. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphol. and physiol. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concns. than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.
    54. 54
      Gerweck, L. E. and Seetharaman, K. (1996) Cellular pH gradient in tumor versus normal tissue: Potential exploitation for the treatment of cancer. Cancer Res. 56, 1194– 1198
      54
      Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer
      Gerweck, Leo E.; Seetharaman, Kala
      Cancer Research (1996), 56 (6), 1194-8CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to primarily represent tissue extracellular pH) is substantially and consistently lower in tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy estd. that intracellular pH is essentially identical or slightly more basic in tumor compared to normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in tumor compared to normal tissue: in normal tissue the extracellular pH is relatively basic, and in tumor tissue the magnitude of the pH gradient is reduced or reversed. This difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized is strongly dependent on the pH of their milieu. Weakly acidic drugs which are relatively lipid sol. in their nonionized state may diffuse freely across the cell membrane and, upon entering a relatively basic intracellular compartment, become trapped and accumulate within a cell, leading to substantial differences in the intracellular/extracellular drug distribution between tumor and normal tissue for drugs exhibiting appropriate pKas.
    55. 55
      Cerutti, P. A. (1985) Prooxidant states and tumor promotion. Science 227, 375– 381,  DOI: 10.1126/science.2981433
      55
      Prooxidant states and tumor promotion
      Cerutti, Peter A.
      Science (Washington, DC, United States) (1985), 227 (4685), 375-81CODEN: SCIEAS; ISSN:0036-8075.
      There is convincing evidence that cellular prooxidant states-i.e., increased concns. of active O and org. peroxides and radicals-can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric O, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P 450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low mol. wt. scavenger mols., and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.
    56. 56
      Bergers, G., Brekken, R., McMahon, G., Vu, T. H., Itoh, T., Tamaki, K., Tanzawa, K., Thorpe, P., Itohara, S., Werb, Z., and Hanahan, D. (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2, 737– 744,  DOI: 10.1038/35036374
      56
      Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis
      Bergers, Gabriele; Brekken, Rolf; McMahon, Gerald; Vu, Thiennu H.; Itoh, Takeshi; Tamaki, Kazuhiko; Tanzawa, Kazuhiko; Thorpe, Philip; Itohara, Shigeyoshi; Werb, Zena; Hanahan, Douglas
      Nature Cell Biology (2000), 2 (10), 737-744CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumor growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumor no. and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumor growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.
    57. 57
      Karimi, M., Ghasemi, A., Sahandi Zangabad, P., Rahighi, R., Moosavi Basri, S. M., Mirshekari, H., Amiri, M., Shafaei Pishabad, Z., Aslani, A., Bozorgomid, M., Ghosh, D., Beyzavi, A., Vaseghi, A., Aref, A. R., Haghani, L., Bahrami, S., and Hamblin, M. R. (2016) Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems. Chem. Soc. Rev. 45, 1457– 1501,  DOI: 10.1039/C5CS00798D
      57
      Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems
      Karimi, Mahdi; Ghasemi, Amir; Sahandi Zangabad, Parham; Rahighi, Reza; Moosavi Basri, S. Masoud; Mirshekari, H.; Amiri, M.; Shafaei Pishabad, Z.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A. R.; Haghani, L.; Bahrami, S.; Hamblin, Michael R.
      Chemical Society Reviews (2016), 45 (5), 1457-1501CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      New achievements in the realm of nanoscience and innovative techniques of nanomedicine have moved micro/nanoparticles (MNPs) to the point of becoming actually useful for practical applications in the near future. Various differences between the extracellular and intracellular environments of cancerous and normal cells and the particular characteristics of tumors such as physicochem. properties, neovasculature, elasticity, surface elec. charge, and pH have motivated the design and fabrication of inventive "smart" MNPs for stimulus-responsive controlled drug release. These novel MNPs can be tailored to be responsive to pH variations, redox potential, enzymic activation, thermal gradients, magnetic fields, light, and ultrasound (US), or can even be responsive to dual or multi-combinations of different stimuli. This unparalleled capability has increased their importance as site-specific controlled drug delivery systems (DDSs) and has encouraged their rapid development in recent years. An in-depth understanding of the underlying mechanisms of these DDS approaches is expected to further contribute to this groundbreaking field of nanomedicine. Smart nanocarriers in the form of MNPs that can be triggered by internal or external stimulus are summarized and discussed in the present review, including pH-sensitive peptides and polymers, redox-responsive micelles and nanogels, thermo- or magnetic-responsive nanoparticles (NPs), mech.- or elec.-responsive MNPs, light or ultrasound-sensitive particles, and multi-responsive MNPs including dual stimuli-sensitive nanosheets of graphene. This review highlights the recent advances of smart MNPs categorized according to their activation stimulus (phys., chem., or biol.) and looks forward to future pharmaceutical applications.
    58. 58
      Zahnd, C., Amstutz, P., and Plückthun, A. (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269– 279,  DOI: 10.1038/nmeth1003
      58
      Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target
      Zahnd, Christian; Amstutz, Patrick; Plueckthun, Andreas
      Nature Methods (2007), 4 (3), 269-279CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Ribosome display is an in vitro selection and evolution technol. for proteins and peptides from large libraries. As it is performed entirely in vitro, there are two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the no. of ribosomes and different mRNA mols. present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations (Box 1). A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide (Fig. 1). The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for anal. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 ext. for in vitro translation, based on the work originally described and further refined in our lab. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display is also included in this issue.
    59. 59
      Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., Ward, C. W., Joos, T. O., and Plückthun, A. (2007) A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J. Mol. Biol. 369, 1015– 28,  DOI: 10.1016/j.jmb.2007.03.028
      59
      A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2
      Zahnd, Christian; Wyler, Emanuel; Schwenk, Jochen M.; Steiner, Daniel; Lawrence, Michael C.; McKern, Neil M.; Pecorari, Frederic; Ward, Colin W.; Joos, Thomas O.; Plueckthun, Andreas
      Journal of Molecular Biology (2007), 369 (4), 1015-1028CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Designed ankyrin repeat proteins (DARPins) are a novel class of binding mols., which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (av. 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma exts. We have detd. the crystal structure of this DARPin at 1.7 Å, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An anal. of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.
    60. 60
      Liu, S., Netzel-Arnett, S., Birkedal-Hansen, H., and Leppla, S. H. (2000) Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin. Cancer Res. 60, 6061– 6067
      60
      Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin
      Liu, Shihui; Netzel-Arnett, Sarah; Birkedal-Hansen, Henning; Leppla, Stephen H.
      Cancer Research (2000), 60 (21), 6061-6067CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed two mutated anthrax toxin protective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by MMPs. These MMP-targeted PA proteins were activated rapidly and selectively on the surface of MMP-overexpressing tumor cells. The activated PA proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A. The toxicity of the mutated PA proteins for MMP-overexpressing cells was blocked by hydroxamate inhibitors of MMPs, including BB94, and by a tissue inhibitor of matrix metalloproteinases (TIMP-2). The mutated PA proteins killed MMP-overexpressing tumor cells while sparing nontumorigenic normal cells when these were grown together in a coculture model, indicating that PA activation occurred on the tumor cell surface and not in the supernatant. This method of achieving cell-type specificity is conceptually distinct from, and potentially synergistic with, the more common strategy of retargeting a protein toxin by fusion to a growth factor, cytokine, or antibody.
    61. 61
      Franke, D., Petoukhov, M. V., Konarev, P. V., Panjkovich, A., Tuukkanen, A., Mertens, H. D. T., Kikhney, A. G., Hajizadeh, N. R., Franklin, J. M., Jeffries, C. M., and Svergun, D. I. (2017) ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212– 1225,  DOI: 10.1107/S1600576717007786
      61
      ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions
      Franke, D.; Petoukhov, M. V.; Konarev, P. V.; Panjkovich, A.; Tuukkanen, A.; Mertens, H. D. T.; Kikhney, A. G.; Hajizadeh, N. R.; Franklin, J. M.; Jeffries, C. M.; Svergun, D. I.
      Journal of Applied Crystallography (2017), 50 (4), 1212-1225CODEN: JACGAR; ISSN:1600-5767. (International Union of Crystallography)
      A review. ATSAS is a comprehensive software suite for the anal. of small-angle scattering data from dil. solns. of biol. macromols. or nanoparticles. It contains applications for primary data processing and assessment, ab initio bead modeling, and model validation, as well as methods for the anal. of flexibility and mixts. In addn., approaches are supported that utilize information from X-ray crystallog., NMR spectroscopy or atomistic homol. modeling to construct hybrid models based on the scattering data. This article summarizes the progress made during the 2.5-2.8 ATSAS release series and highlights the latest developments. These include AMBIMETER, an assessment of the reconstruction ambiguity of exptl. data; DATCLASS, a multiclass shape classification based on exptl. data; SASRES, for estg. the resoln. of ab initio model reconstructions; CHROMIXS, a convenient interface to analyze in-line size exclusion chromatog. data; SHANUM, to evaluate the useful angular range in measured data; SREFLEX, to refine available high-resoln. models using normal mode anal.; SUPALM for a rapid superposition of low- and high-resoln. models; and SASPy, the ATSAS plugin for interactive modeling in PyMOL. All these features and other improvements are included in the ATSAS release 2.8, freely available for academic users from https://www.embl-hamburg.de/biosaxs/software.html.</a></a></a></a></a></a></a></a></a>.
    62. 62
      Franke, D. and Svergun, D. I. (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342– 346,  DOI: 10.1107/S0021889809000338
      62
      DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering
      Franke, Daniel; Svergun, Dmitri I.
      Journal of Applied Crystallography (2009), 42 (2), 342-346CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
      DAMMIF, a revised implementation of the ab-initio shape-detn. program DAMMIN for small-angle scattering data, is presented. The program was fully rewritten, and its algorithm was optimized for speed of execution and modified to avoid limitations due to the finite search vol. Symmetry and anisometry constraints can be imposed on the particle shape, similar to DAMMIN. In equivalent conditions, DAMMIF is 25-40 times faster than DAMMIN on a single CPU. The possibility to utilize multiple CPUs is added to DAMMIF. The application is available in binary form for major platforms.
    63. 63
      Volkov, V. V. and Svergun, D. I. (2003) Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860– 864,  DOI: 10.1107/S0021889803000268
      63
      Uniqueness of ab initio shape determination in small-angle scattering
      Volkov, Vladimir V.; Svergun, Dmitri I.
      Journal of Applied Crystallography (2003), 36 (3, Pt. 1), 860-864CODEN: JACGAR; ISSN:0021-8898. (Blackwell Munksgaard)
      Scattering patterns from geometrical bodies with different shapes and anisometry (solid and hollow spheres, cylinders, prisms) are computed and the shapes are reconstructed ab initio using envelope function and bead modeling methods. A procedure is described to analyze multiple solns. provided by bead modeling methods and to est. stability and reliability of the shape reconstruction. It is demonstrated that flat shapes are more difficult to restore than elongated ones and types of shapes are indicated, which require addnl. information for reliable shape reconstruction from the scattering data.
    64. 64
      The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC.
      There is no corresponding record for this reference.
    65. 65
      Webb, B., and Sali, A. (2017) Protein Structure Modeling with MODELLER, in Functional Genomics: Methods and Protocols, Methods in Molecular Biology, (Kaufmann, M., Klinger, C., and Savelsbergh, A., Eds.), pp 39– 54, Humana Press, New York, NY.
      There is no corresponding record for this reference.
    66. 66
      Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., De Beer, T. A. P., Rempfer, C., Bordoli, L., Lepore, R., and Schwede, T. (2018) SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296– W303,  DOI: 10.1093/nar/gky427
      66
      SWISS-MODEL: homology modelling of protein structures and complexes
      Waterhouse, Andrew; Bertoni, Martino; Bienert, Stefan; Studer, Gabriel; Tauriello, Gerardo; Gumienny, Rafal; Heer, Florian T.; de Beer, Tjaart A. P.; Rempfer, Christine; Bordoli, Lorenza; Lepore, Rosalba; Schwede, Torsten
      Nucleic Acids Research (2018), 46 (W1), W296-W303CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Homol. modeling has matured into an important technique in structural biol., significantly contributing to narrowing the gap between known protein sequences and exptl. detd. structures. Fully automated workflows and servers simplify and streamline the homol. modeling process, also allowing users without a specific computational expertise to generate reliable protein models and have easy access to modeling results, their visualization and interpretation. Here, we present an update to the SWISS-MODEL server, which pioneered the field of automated modeling 25 years ago and been continuously further developed. Recently, its functionality has been extended to the modeling of homo- and heteromeric complexes. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex are inferred by homol. modeling. Other major improvements include the implementation of a new modeling engine, ProMod3 and the introduction a new local model quality estn. method, QMEANDisCo.
    67. 67
      Schweizer, A., Roschitzki-Voser, H., Amstutz, P., Briand, C., Gulotti-Georgieva, M., Prenosil, E., Binz, H. K., Capitani, G., Baici, A., Plückthun, A., and Grütter, M. G. (2007) Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. Structure 15, 625– 36,  DOI: 10.1016/j.str.2007.03.014
      67
      Inhibition of Caspase-2 by a Designed Ankyrin Repeat Protein: Specificity, Structure, and Inhibition Mechanism
      Schweizer, Andreas; Roschitzki-Voser, Heidi; Amstutz, Patrick; Briand, Christophe; Gulotti-Georgieva, Maya; Prenosil, Eva; Binz, H. Kaspar; Capitani, Guido; Baici, Antonio; Plueckthun, Andreas; Gruetter, Markus G.
      Structure (Cambridge, MA, United States) (2007), 15 (5), 625-636CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Summary: Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition const. and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the mol. basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biol. apoptotic signaling network.
    68. 68
      Petoukhov, M. V. and Svergun, D. I. (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys. J. 89, 1237– 1250,  DOI: 10.1529/biophysj.105.064154
      68
      Global rigid body modeling of macromolecular complexes against small-angle scattering data
      Petoukhov, Maxim V.; Svergun, Dmitri I.
      Biophysical Journal (2005), 89 (2), 1237-1250CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      New methods to automatically build models of macromol. complexes from high-resoln. structures or homol. models of their subunits or domains against x-ray or neutron small-angle scattering data are presented. Depending on the complexity of the object, different approaches are employed for the global search of the optimum configuration of subunits fitting the exptl. data. An exhaustive grid search is used for hetero- and homodimeric particles and for sym. oligomers formed by identical subunits. For the assemblies or multidomain proteins contg. more then one subunit/domain per asym. unit, heuristic algorithms based on simulated annealing are used. Fast computational algorithms based on spherical harmonics representation of scattering amplitudes are employed. The methods allow one to construct interconnected models without steric clashes, to account for the particle symmetry and to incorporate information from other methods, on distances between specific residues or nucleotides. For multidomain proteins, addn. of missing linkers between the domains is possible. Simultaneous fitting of multiple scattering patterns from subcomplexes or deletion mutants is incorporated. The efficiency of the methods is illustrated by their application to complexes of different types in several simulated and practical examples. Limitations and possible ambiguity of rigid body modeling are discussed and simplified docking criteria are provided to rank multiple models. The methods described are implemented in publicly available computer programs running on major hardware platforms.
    69. 69
      Panjkovich, A. and Svergun, D. I. (2016) SASpy: A PyMOL plugin for manipulation and refinement of hybrid models against small angle X-ray scattering data. Bioinformatics 32, 2062– 2064,  DOI: 10.1093/bioinformatics/btw071
      69
      SASpy: a PyMOL plugin for manipulation and refinement of hybrid models against small angle X-ray scattering data
      Panjkovich, Alejandro; Svergun, Dmitri I.
      Bioinformatics (2016), 32 (13), 2062-2064CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
      Complex formation and conformational transitions of biol. macromols. in soln. can be effectively studied using the information about overall shape and size provided by small angle X-ray scattering (SAXS). Hybrid modeling is often applied to integrate high-resoln. models into SAXS data anal. To facilitate this task, we present SASpy, a PyMOL plugin that provides an easy-to-use graphical interface for SAXS-based hybrid modeling. Through a few mouse clicks in SASpy, low-resoln. models can be superimposed to high-resoln. structures, theor. scattering profiles and fits can be calcd. and displayed on-the-fly. Mouse-based manual rearrangements of complexes are conveniently applied to rapidly check and interactively refine tentative models. Interfaces to automated rigid-body and flexible refinement of macromol. models against the exptl. SAXS data are provided.
    70. 70
      Hodnik, V., and Anderluh, G. (2013) Surface Plasmon Resonance for Measuring Interactions of Proteins with Lipid Membranes, in Lipid-Protein Interactions: Methods and Protocols, Methods in Molecular Biology, (Kleinschmidt, J. H., Ed.), pp 23– 36, Humana Press, Totowa, NJ.
      There is no corresponding record for this reference.
    71. 71
      Anderluh, G., Beseničar, M., Kladnik, A., Lakey, J. H., and Maček, P. (2005) Properties of nonfused liposomes immobilized on an L1 Biacore chip and their permeabilization by a eukaryotic pore-forming toxin. Anal. Biochem. 344, 43– 52,  DOI: 10.1016/j.ab.2005.06.013
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      Properties of nonfused liposomes immobilized on an L1 Biacore chip and their permeabilization by a eukaryotic pore-forming toxin
      Anderluh, Gregor; Besenicar, Mojca; Kladnik, Ales; Lakey, Jeremy H.; Macek, Peter
      Analytical Biochemistry (2005), 344 (1), 43-52CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      The L1 chip is used intensively for protein-membrane interaction studies in Biacore surface plasmon resonance systems. The exact form of captured lipid membranes on the chip is, however, not precisely known. Evidence exists that the vesicles both remain intact after the binding to the chip and fuse to form a large single-bilayer membrane. In this study, the authors were able to bind up to approx. 11,500 resonance units of zwitterionic liposomes (100 nm in diam.) at a low flow rate. The authors show by fluorescence microscopy that the entire surface of the flow cell is covered homogeneously by liposomes. Neg. charged vesicles (i.e., those composed of phosphatidylcholine/phosphatidylglycerol [1:1]) always deposited less densely, but the authors were able to increase the d. slightly with the use of calcium chloride that promotes fusion of the vesicles. Finally, the authors used zwitterionic liposomes loaded with fluorescent probe calcein to show that they remain intact after the capture on the L1 chip. The fluorescence was lost only after the authors used equinatoxin, a well-studied pore-forming toxin, to perform on-chip permeabilization of vesicles. The characteristics of permeabilization process for chip-immobilized liposomes are similar to those of liposomes free in soln. All results collectively suggest that liposomes do not fuse to form a single bilayer on the surface of the chip.
    72. 72
      Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671– 675,  DOI: 10.1038/nmeth.2089
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      NIH Image to ImageJ: 25 years of image analysis
      Schneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W.
      Nature Methods (2012), 9 (7_part1), 671-675CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the anal. of scientific images. We discuss the origins, challenges and solns. of these two programs, and how their history can serve to advise and inform other software projects.