Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Am. Chem. Soc. 2013, 135, 49, 18673-18681
Naturally Split Inteins Assemble through a “Capture and Collapse” Mechanism
View Author Information
† Department of Chemistry, Princeton University, Frick Laboratory, Princeton, New Jersey 08544, United States
‡ Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States
Copyright © 2013 American Chemical Society
Abstract
Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.
Introduction
ARTICLE SECTIONS
Inteins are autoprocessing domains that facilitate their own excision from larger precursor proteins while ligating their flanking polypeptide sequences (exteins) through a native peptide bond.(1) This process, known as protein splicing, occurs in unicellular organisms from all domains of life.(2, 3) The chemical mechanism of protein splicing is well-understood, and this has led to the development of numerous protein engineering tools that harness inteins to chemically manipulate virtually any protein of interest.(4) There are two types of naturally occurring inteins: highly abundant contiguous inteins and rarer split inteins. The latter carry out protein splicing in trans, where the biochemistry is preceded by the spontaneous association of two separately transcribed and translated fragments (N- and C-inteins, Figure 1a). Although naturally split inteins evolved from their contiguous counterparts and share the same fold,(5, 6) it is notable that many artificially split versions of contiguous inteins cannot spontaneously assemble and splice.(7-9) The intein fold has a complex interwoven topology (Figure 1b),(10) and split inteins must bear unique features that allow them to access this topology in trans. Given that inteins splice essential proteins, there would have been strong selective pressure to optimize fragment complementation in early split inteins. Indeed, this evolution is evident in modern split inteins, which associate rapidly and tightly.(11-13)
Figure 1
Figure 1. Characterization of Npu fragments and complex. (a) Scheme depicting the genetic origin of split intein fragments and their role in protein trans-splicing. (b) Rendering of the Npu intein structure, highlighting relevant NpuN lobes (NpuN1 in dark blue and NpuN2 in light blue) and NpuC in red. Termini to which exteins would be attached are shown as orange spheres, and sites of intrinsic fluorophores used in this study (W47 and Y58W) are shown as green sticks; rendering based on PDB 2KEQ.(10) (c) SEC-MALS of NpuN (blue, 25 μM, expected MW = 12188 Da), NpuC (red, 400 μM, expected MW = 4443 Da), and their complex (black, 25 μM, expected MW = 16631 Da). (d) 1H–15N HSQC spectra of NpuC alone (red, 250 μM, 600 MHz) and in complex with unlabeled NpuN (black, 250 μM, 800 MHz). (e) 1H–15N HSQC spectra of NpuN alone (blue, 250 μM, 500 MHz) and in complex with unlabeled NpuC (black, 250 μM, 800 MHz). NMR spectra were acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
The largest known class of split inteins is the cyanobacterial DnaE intein family.(5) In their natural context, these inteins assemble the catalytic subunit of DNA polymerase III (DnaE). Several members of this family carry out protein splicing with remarkable efficiency, in tens of seconds rather than hours as is the case for many other inteins, making them ideal tools for protein engineering.(14, 15) Of these ultrafast trans-splicing domains, the DnaE intein from Nostoc punctiforme (Npu) is the best-characterized member, and it is rapidly becoming the intein of choice for new protein chemistry technologies.(16-19) Currently, little is known about the assembly of Npu or other split DnaE inteins. We previously demonstrated that the Npu fragments associate with low nanomolar affinity,(12) similar to orthologous split intein fragments from Synechocystis sp. PCC6803 (Ssp).(11, 13) Atomic force microscopy measurements on three other split DnaE inteins suggest that this tight binding is a conserved property in this family.(20) Furthermore, Ssp binding is known to be extraordinarily fast and ionic strength dependent.(11) Recently, it was proposed that both Ssp fragments undergo a disorder-to-order transition upon binding; however, the molecular determinants and mechanism of this transition were not established.(13) On the basis of sequence analyses, it is clear that most split inteins differ from contiguous ones by the presence of significant charge segregation between the N- and C-inteins (Figure S1), and indeed, mutating critical ion pairs and triads at the fragment interface weakens fragment affinity.(12)
Here we elucidate the mechanism of split intein assembly, using Npu as a relevant model system. We show that split intein fragments interact through a multiphase process initiated by electrostatic interactions between extended regions of both fragments (capture) followed by compaction and stabilization of the initially disordered segments onto a prefolded region of the N-intein (collapse). Given the emerging role of split inteins as powerful tools for in vitro and in vivo protein engineering and semisynthesis,(21) this biophysical mechanism should aid in the development and further improvement of split intein-based technologies.
Results
ARTICLE SECTIONS
Npu Fragments Undergo Dramatic Conformational Changes upon Association
To elucidate the binding mechanism of Npu and related inteins, we initially sought to characterize the structures of the 102 residue N-intein (NpuN) and 35 residue C-intein (NpuC) in isolation. Both proteins were generated and purified as previously described, bearing short tripeptide exteins that should not contribute significantly to the spectroscopic or chromatographic signals being observed (Figures S2 and S3).(22) These constructs also contained a C1A mutation in the N-intein and N137A mutation in the C-intein to prevent protein splicing during analyses of the split intein complex.(22) Circular dichroism spectra of the fragments and an equimolar mixture suggested low α and β secondary structure content in the isolated fragments and a dramatic structural change upon binding (Figure S4). Consistent with these data, NpuC was extremely susceptible to degradation by thermolysin, whereas the complex was effectively resistant to proteolysis (Figure S5a,b). NpuN showed an intermediate rate of degradation, suggesting a partially compact structure (Figure S5c). Remarkably, the N-intein eluted earlier than the complex on a size exclusion chromatography (SEC) column, indicating that it must collapse from an extended state upon binding NpuC (Figure 1c). To rule out the possibility of dimerization, we performed multiangle light scattering (MALS) online with SEC and confirmed that NpuN is a monomer in solution (Figure 1c).
Additional insight into fragment structure was gained from nuclear magnetic resonance (NMR) spectroscopy. The 1H–15N HSQC spectrum of uniformly 15N-labeled NpuC showed sharp amide N–H resonances with poor dispersion along the 1H axis, indicative of an unfolded protein (Figure 1d, red). Notably, a similar spectrum was previously observed for the homologous Ssp C-intein, suggesting conserved structural properties among split inteins, even in the unbound state.(13) Upon addition of unlabeled NpuN, the signal dispersion increased dramatically, consistent with NpuC being part of a well-folded protein domain (Figure 1d, black). In the bound and unbound states, backbone resonances were readily assigned to aid in further analyses. NMR relaxation experiments (R1, R2, and heteronuclear NOE measurements) on isolated NpuC confirmed the high degree of flexibility throughout the chain with no detectable residual secondary structure (Figure S6). In contrast to NpuC, the HSQC spectrum of isolated NpuN showed moderate signal dispersion, however only a fraction of the expected resonances were visible, and the observed signals varied greatly in relative intensity (Figure 1e, blue). These data depict an N-intein that is highly dynamic but at least partially folded, consistent with both the expanded dimensions observed by SEC and the intermediate protection from thermolysin proteolysis. While these experiments provide a general picture of NpuN, the nature of the NMR data precluded backbone resonance assignments and thus sequence-specific information.
NpuN Is Comprised of Two Structurally Distinct Lobes
We previously demonstrated that Npu fragment binding is dependent on electrostatic interactions between the highly acidic NpuN (calculated pI = 4.4) and highly basic NpuC (calculated pI = 9.7).(12) The majority of relevant anionic residues on NpuN are concentrated to the second half of the sequence (NpuN2, residues 51–102), which is spatially distinct from the first half of the sequence (NpuN1, residues 1–50) in the final intein complex (Figures 1b and S1, light and dark blue regions, respectively). This uneven distribution of charges is clearly seen in the final complex, where the interface between NpuN1 and NpuC is primarily hydrophobic (Figure 2a,b) while NpuN2 and NpuC have significant electrostatic complementarity (Figure 2c,d). Given these observations, we hypothesized that the different electrostatic properties of these two NpuN lobes may shed light on the overall structure of NpuN in isolation. Indeed, a closer analysis of the sequence composition of these lobes indicated that the first lobe, NpuN1, has a high density of hydrophobic residues and low net charge per residue, consistent with a well-folded protein (Figure 3a). By contrast, the second lobe, NpuN2, has a high net charge per residue and lower hydrophobicity, a hallmark of intrinsically disordered proteins (IDPs, Figure 3a).(23) Furthermore, despite their virtually identical molecular weights (ΔMW = 2 Da) and their symmetry-related structures in the final complex (Figure 1b), isolated NpuN1 eluted later than NpuN2 on a SEC column, suggesting a more compact structure for this lobe (Figures 3b and S7a–h). The more expanded lobe, NpuN2, could be compacted in the presence of high salt, a known property of IDPs (Figure S7i).(24) Additionally, NpuN1, but not NpuN2, bound the hydrophobic dye 1-anilino-8-naphthalene sulfonate (ANS), suggesting that this compact lobe has molten globule-like properties with solvent-exposed hydrophobic surfaces (Figure S7j).(25) As discussed above, the interface between NpuN1 and NpuC in the final split intein complex is dominated by hydrophobic interactions (Figure 2), which presumably satisfy these exposed patches.
Figure 2
Figure 2. Electrostatic surface representations of Npu. The color scheme for these representations is red for negative charge, white for neutral, and blue for positive charge. All perspectives are given relative to panel (a), which is the same perspective as Figure 1b. The renderings are based on PDB 2KEQ.(10) Panels a and b highlight complementary hydrophobic surfaces on either fragment at the NpuN1–NpuC interface. Panels c and d highlight complementary electrostatic surfaces on either fragment at the NpuN2–NpuC interface.
Figure 3
Figure 3. Characterization of the NpuN lobes. (a) Charge-hydrophobicity plot comparing Npu fragments, complex, and N-intein lobes. Mean hydrophobicity (H) is calculated on a normalized Kyte-Doolittle scale, and mean net charge (R) is the absolute value. The solid line delineating disordered and folded proteins is empirically defined as R = 2.785·H – 1.151.(23) (b) SEC of NpuN1 (dark blue, 7 μM), NpuN2 (light blue, 7 μM) and NpuC (red, 7 μM). (c) Scheme depicting the segmental isotopic labeling of NpuN using Expressed Protein Ligation. (d) 1H–15N HSQC spectra of NpuN segmentally 15N-labeled on NpuN1 (dark blue, 100 μM) and NpuN2 (light blue, 100 μM) collected at 500 MHz. NMR data was acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
We next asked if the different structural properties observed in the isolated NpuN lobes are retained in the context of full-length NpuN. To address this, we identified products from the limited proteolysis of NpuN by mass spectrometry. Early in the proteolysis reaction, we identified several pairs of products consistent with single proteolytic events (Figure S5d–f). As expected, in full-length NpuN, the second lobe was more susceptible to proteolysis than the first, supporting the notion that it is less compact. To more definitively assess the relative lobe structures, we employed Expressed Protein Ligation (EPL)(26) to generate full-length NpuN with segmental isotope labeling either on NpuN1 or NpuN2. Residue 59 in NpuN, eight residues downstream of the NpuN1–NpuN2 junction, is a native cysteine and provides a reactive handle for ligation of the lobes while retaining the wild-type NpuN sequence (Figure 3c and S8). This ligation approach allows for the selective observation of either NpuN1 or NpuN2 resonances in the context of full-length NpuN. The 1H–15N HSQC spectrum of the NpuN1-labeled sample showed markedly greater proton chemical shift dispersion than that of the NpuN2-labeled sample (Figure 3d). These spectra confirm the bipartite structure of the N-intein, with an at least partially folded NpuN1 lobe and a disordered NpuN2 lobe. Interestingly, many of the NpuN1 resonances that were visible on a 500 MHz spectrometer were not visible at 800 MHz due to chemical exchange line-broadening, whereas NpuN2 resonances were less affected by the field strength of the spectrometer (Figure S9). Thus, the two connected lobes have different chemical exchange rates indicating internal dynamics on different time scales,(27) consistent with their distinct states of compaction. Importantly, both segmentally labeled N-inteins could bind NpuC to yield correctly folded, functional complexes (Figure S10).
The NpuN2–NpuC Interaction Represents a Binding Intermediate
Given their distinct structures, we hypothesized that NpuN1 and NpuN2 play unique roles in engaging NpuC. To test this, we analyzed mixtures of each isolated lobe with NpuC by SEC-MALS at concentrations ranging from 7 to 50 μM. No interaction was observed between the two N-intein lobes or between NpuN1 and NpuC at any concentration tested (Figure S11a–c). Remarkably, a 1:1 complex between NpuN2 and NpuC was unambiguously observed by SEC-MALS (Figures 4a and S11d). Notably, this complex eluted later than NpuN2 alone, indicating that this lobe is compacted upon binding the C-intein (Figures 4a and S7f). Furthermore, binding was weakened by increasing the ionic strength, demonstrating that there is an electrostatic component to this interaction (Figure S11e,f).
Figure 4
Figure 4. Characterization of the NpuN2–NpuC interaction. (a) SEC-MALS of isolated NpuN2 (light blue, 62.5 μM, expected MW = 6098 Da), NpuC (red, 7 μM), and an equimolar mixture of NpuN2 and NpuC (black, 50 μM, expected MW = 10541 Da). (b) 1H–15N HSQC spectra of NpuN2 alone (light blue, 250 μM) and with equimolar unlabeled NpuC (black, 250 μM) collected at 800 MHz. (c) Chemical shift index values (ΔC/H = δC/HObserved – δC/HRandomCoil) for Cα, CO, and HN atoms for NpuN2 in complex with NpuC. The consensus secondary structure prediction is shown below compared with NpuN2 in the context of the full Npu complex (as seen in PDB 2KEQ).(10) The NpuN2 sequence is given above, with important anionic residues in orange. (d) Composite 1H and 15N backbone chemical shift perturbation values for NpuC in complex with NpuN (black) or NpuN2 (light blue) calculated relative to isolated NpuC. Δδi = [(ΔδH,complex – ΔδH,alone)i2 + 0.11(ΔδN,complex – ΔδN,alone)i2 ]1/2 The NpuC sequence is given above, with important cationic residues in orange. Below the sequence, an asterisk marks unassigned residues for isolated NpuC (red), the complex with NpuN (black), or the complex with NpuN2 (light blue). (e) Rendering of the NpuN2–NpuC interaction, as seen in the native Npu complex (PDB 2KEQ).(10) NpuN2 is light blue, NpuC is red, and charged residues involved in intermolecular electrostatic interactions are highlighted as orange sticks. Predicted β-sheet and α-helical regions of NpuN2 are colored in green and purple, respectively. Residues 103–115 of NpuC are rendered as a ribbon, and the remainder of the sequence is rendered as a dotted line. (f) 1H–15N HSQC spectrum of Npu(51–115) collected at 600 MHz. NMR data in panels b, c, d, and f were acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
Since NpuC preferentially binds NpuN2 over NpuN1, we postulate that the NpuN2–NpuC complex represents an intermediate in the coupled binding and folding pathway for DnaE inteins. Thus, we sought to characterize the structure of this complex. The 1H–15N HSQC spectrum of isolated NpuN2 in the absence and presence of NpuC suggested a global structural transition from a highly flexible state to a collapsed state (Figure 4b). On the basis of their deviation from average random coil values, the backbone 1H and 13C chemical shifts for NpuN2 both alone and in complex with NpuC were used to predict secondary structure content in this lobe (Figures 4c and S12a–d). In the absence of NpuC, NpuN2 had no predicted secondary structural elements, consistent with it being an IDP. In the presence of NpuC, however, NpuN2 was predicted to have α-helical and β-sheet content in the same regions as in the native Npu complex, particularly for residues at the interface with NpuC (Figure 4c).
Unlike the global conformational change seen for NpuN2 upon binding NpuC, only a fraction of the NpuC backbone resonances showed chemical shift perturbations upon binding NpuN2, while the remainder retained the chemical shifts seen for isolated NpuC (Figure S12e,f). Notably, for those resonances that changed, the magnitude and direction of the chemical shift perturbation was similar to that seen for the same residues in full-length complex, suggesting native-like structure at these positions (Figures 4d and S12f). The perturbed residues primarily correspond to a contiguous stretch of nine amino acids at the N-terminus of NpuC (104–112), which make both ionic and β-strand pairing interactions with NpuN2 in the full-length complex (Figure 4e). To further confirm that these C-intein residues were important for NpuN2 binding and collapse, we generated a fusion between NpuN2 and the first 13 residues of NpuC, NpuN(51–115). As expected, the 1H–15N HSQC spectrum of Npu(51–115) described a well-folded protein, indicating that this short stretch of NpuC was sufficient to collapse NpuN2 (Figure 4f).
Split Intein Assembly Is Multiphasic and Electrostatically Driven
The presence of a well-defined binding intermediate such as the NpuN2–NpuC complex indicates that split intein assembly is multiphasic and that these phases may be discretely observable. The fluorescence of the sole tryptophan in the Npu complex (W47, Figure 1b) is quenched upon Npu assembly and can be used to monitor binding (Figure 5a).(12) Using this probe, we measured an equilibrium dissociation constant (KD) for Npu of 1.2 ± 0.8 nM (Figure S13). In kinetic measurements using stopped-flow fluorescence, only a single binding phase was observable with a kon of (8.9 ± 0.3) × 105 M–1 s–1 (Figures 5b,c and S14a) and an off-rate too slow to extract directly from our kinetic data (when calculated from KD and kon, the koff is between 10–4 and 10–3 s–1). W47 lies in NpuN1, which our data indicate is compact throughout split intein assembly and may not be sensitive to multiple steps in the binding process. Thus, we engineered a new tryptophan in the more dynamic NpuN2 (Y58W, Figure 1b) and mutated W47 to tyrosine to silence NpuN1 fluorescence. These mutations modestly affected the binding equilibrium (KD = 3.4 ± 1.0 nM, Figure S13) and had no effect on splicing activity (Figure S15). Unlike W47 fluorescence, W58 fluorescence increased dramatically upon fragment association, consistent with NpuN2 undergoing a folding transition upon binding NpuC (Figure 5d). Stopped-flow fluorescence of this mutant divulged two binding phases (Figures 5e,f and S14b,c), an initial concentration-dependent fast phase with a kon of (2.7 ± 0.4) × 105 M–1 s–1 followed by a slower concentration-independent phase (kobs = 0.04 ± 0.02 s–1) that was only marginally faster than the overall protein splicing rate (ksplice = ∼0.01 s–1, Figure S15). Notably, for both the wild-type and mutant inteins, the KD increased and kon decreased in higher ionic strength buffers (Figures S13 and S14).
Figure 5
Figure 5. Intrinsic fluorescence binding measurements: (a) Tryptophan fluorescence of wild-type NpuN in the absence and presence of NpuC (λex = 290 nm); (b) Tryptophan fluorescence of wild-type Npu upon stopped-flow mixing of fragments (λex = 290 nm, λem = >320 nm); (c) Residuals from fits to a one-phase (top) and two-phase (bottom) binding model for wild-type Npu; (d) Tryptophan fluorescence of NpuN with the W47Y and Y58W mutations in the absence and presence of NpuC (λex = 290 nm); (e) Tryptophan fluorescence of Npu W47Y,Y58W upon stopped-flow mixing of fragments (λex = 290 nm, λem = >320 nm); (f) Residuals from fits to a one-phase (top) and two-phase (bottom) binding model for Npu W47Y,Y58W.
Our fluorescence binding measurements are consistent with a rapid, electrostatically driven encounter between two fragments followed by a collapse of the structure. As discussed in the preceding sections, the electrostatic component to binding most likely originates from the interaction between NpuN2 and NpuC. In a previous study, we mutated residues involved in several intermolecular ionic interactions throughout the fragment interface in Npu, swapping anionic residues in NpuN for cationic ones and cationic residues in NpuC for anionic ones.(12) We found that introducing repulsive electrostatic interactions at the fragment interface often diminished splicing activity, and compensatory electrostatic mutations typically recovered activity. Importantly, this effect on activity correlated with binding affinity. Furthermore, the region of the intein most sensitive to electrostatic mutations comprised an ion triad on the face of the α-helix at the core of the NpuN2–NpuC interface (D85, E89, and R108), consistent with the biophysical data presented herein.
Split Intein Fragments Associate via a “Capture and Collapse” Mechanism
On the basis of the foregoing biophysical experiments, we propose that Npu fragment assembly is a multistep process in which NpuC is first engaged by NpuN2, followed by NpuN1 (Figure 6). The initial encounter between the fragments is dictated by electrostatic interactions between the disordered, highly cationic NpuC and the extended, highly anionic second lobe of NpuN. This encounter complex resolves to an intermediate that has secondary structure and intermolecular interactions resembling half of the native Npu complex. After this initial “capture”, the flexible regions “collapse” into an ordered state that is further stabilized by hydrophobic interactions between NpuC and the well-ordered first lobe of NpuN. The resulting complex has intertwined fragments, and this topology is presumably only accessible due to the intrinsic disorder of the starting materials. This “capture and collapse” model is meant to provide a framework for understanding intein fragment assembly, and we recognize that there could be additional folding steps within each phase that are not detectable using the biophysical approaches employed herein.
Figure 6
Figure 6. The “capture and collapse” mechanism of split intein assembly. An N-terminal segment of the disordered C-intein is captured by the extended second lobe of the N-intein with compaction of that lobe into a native-like structure. This intermediate then collapses as the remainder of the C-intein docks into the preorganized first lobe of the N-intein.
Native Topology Is Important for Split Intein Stability and Function
NpuC intercalates between the NpuN lobes, allowing it to engage NpuN2 through electrostatic interactions and NpuN1 through hydrophobic interactions (Figure 2). These segregated intermolecular interactions and the resulting interweaving of the chains should not only drive assembly, as described above, but also prevent the bound fragments from rapidly dissociating, thereby precluding splicing. To test this notion, we engineered two Npu variants with altered primary sequence topology. In the first system, we permuted the split intein sequence to yield two new proteins related by a pseudosymmetry axis (Figures 7a and S2). One protein, Npu(51–115) described above represents the well-folded binding intermediate composed of NpuN2 and a segment of NpuC, where all crucial ionic interactions are presatisfied (Figure 4e,f). The second protein, a fusion of NpuN1 and the remainder of NpuC, presatisfies the hydrophobic interactions between these two protomers (Figure 2a,b). A complex of these two permuted fragments should associate slowly and dissociate rapidly, given the lack of key intermolecular interactions and entwined fragments. As expected, the permuted intein fragments could assemble into the correct intein fold, as evidenced by their capacity to carry out protein splicing when fused to model extein domains, ubiquitin (Ub) and SUMO (Figures 7b,c and S16). However, the rate of splicing was extremely concentration dependent, indicating that association is rate-limiting. It is noteworthy that, even at high concentrations, splicing was dramatically slower for this permuted construct than wild-type Npu (days instead of minutes). As the complex was too unstable to measure binding parameters, we attribute this low efficiency at high concentrations to a short lifetime of the active complex caused by a fast off-rate.
Figure 7
Figure 7. Split intein assembly and function with engineered sequence topologies. (a) Structural representation of the two intein regions in the permuted Npu constructs (based on PDB 2KEQ).(10) The dashed line represents a diglycine linker introduced between residues 1–50 and 116–137. (b) Scheme showing protein splicing with the permuted Npu. Ub (ubiquitin) and SUMO are model N- and C-exteins. (c) Proteins splicing of permuted Npu at 10 and 100 μM fragment concentrations. Reaction mixtures when analyzed at indicated time-points by SDS-PAGE with coomassie staining. (d) SEC-MALS of the three-piece Npu complex (black, 7 μM of each fragment, expected MW = 16649 Da) compared with the individual components (NpuN1, dark blue; NpuN2, light blue; NpuC, red; each at 7 μM). (e) Scheme showing protein splicing with the three-piece Npu. (f) Splicing activity of the three-piece Npu at 5 μM fragment concentrations. Reaction mixtures where analyzed at indicated time-points by SDS-PAGE with coomassie staining. The asterisk indicates a side product, most likely SUMO due to premature N-extein cleavage.
In the second system, we utilized the isolated NpuN lobes in conjunction with NpuC to generate a three-piece intein. While these constructs must contend with the entropic cost of forming a ternary complex, the key intermolecular interactions between each lobe and NpuC are still present. The three-piece complex was stable at low-micromolar concentrations (Figure 7d) where the binary interaction between NpuN2 and NpuC was barely visible (Figure S11d). Since NpuC cannot independently interact with NpuN1 (Figure S11b,c), this result shows that the lobes bind NpuC cooperatively. Notably, the resulting ternary complex could carry out protein splicing in the context of model SUMO extein domains with substantially higher efficiency than the permuted complex (Figures 7e,f and S17), demonstrating the kinetic stability of the active complex.
Discussion and Conclusions
ARTICLE SECTIONS
Inteins have a unique fold in which the polypeptide chain traverses back and forth between two symmetry-related halves (Figure 1b). Given this, it is remarkable that some inteins are naturally split, as this topology precludes a binding mechanism involving natively structured fragments. In this report, we describe the structure of split intein fragments and determine their mechanism of association. Using a variety of biophysical techniques coupled with protein engineering and protein chemistry, we show that the N-intein fragment has a unique bipartite structure comprised of one at least partially folded lobe tethered to an intrinsically disordered region of equal length. This extended region of the N-intein captures the completely disordered C-intein through electrostatic interactions, leading to a native-like intermediate. This intermediate then further collapses onto the more structured N-intein lobe, resulting in a functional intein domain with entangled fragments.
To function, split intein fragments must not only find one another efficiently, but also bind to form a stable complex that can persist throughout the duration of the complex multistep splicing reaction, which can last from seconds to hours.(15) Our studies indicate that split DnaE inteins have evolved several unique structural features that fulfill these requirements: (1) The initial encounter complex is dictated by two disordered protein regions, which should provide larger capture radii for an enhanced collision probability.(28) (2) This enhanced on-rate is further reinforced by electrostatic steering of these fragments.(29) (3) The slow off-rate is presumably governed by the retention of these ionic interactions in the final complex (Figure 2c,d),(12) as well as significant hydrophobic interactions between NpuC and NpuN1 (Figure 2a,b). (4) The disordered regions of the fragments provide access to an interwoven topology, which further reduces the off-rate (Figure 1b).
The significance of this interweaving for intein function was demonstrated using two engineered systems: one with a permuted primary sequence and another involving a three-piece intein. It is noteworthy that both of these systems could, in principle, be used to design novel intein-based technologies. The permuted system, with poor binding, could be enhanced by fusion to an inducible dimerization system, thus making a conditionally splicing Npu. To our knowledge, such a system has never been developed with a highly efficient intein such as Npu, as the native fragments of this and other naturally split inteins spontaneously associate. Instead, conditional protein splicing (CPS) modules have traditionally been developed using less efficient artificially split inteins, which require fusion to dimerization domains as they typically lack the key properties required to function well in trans: charge complementarity and well-behaved disordered fragments. Construction of a controllable fast-splicing Npu intein would be extremely desirable, as CPS systems show promise as tools to control protein function in vivo in response to external stimuli.(9) The three-piece system is particularly intriguing, as it is implicitly conditional: the central piece, NpuN2, is effectively a trigger for protein oligomerization and splicing. Further design and optimization of both the permuted and three-piece Npu inteins should lead to the development of useful tools for biochemistry and cell biology.
Split inteins likely arose from their contiguous predecessors due to DNA inversions or aberrant gene insertions and excisions.(5, 6) These genomic rearrangements independently occurred several times, as evidenced by the diversity of split inteins, their host proteins, and their host organisms.(5, 6, 30) Interestingly, even outside of the DnaE family described in this study, many split inteins are cut at the same point on the intein fold and exhibit dramatic charge segregation between fragments (Figure S1c). Furthermore, homology modeling of non-DnaE split inteins predicts that many have intermolecular electrostatic interactions at the ridge of their horseshoe-like structure, as seen in the DnaE inteins.(6) Thus, the efficient folding mechanism for Npu is most likely a general solution at the protein level to the rare but repeated fracturing of intein genes at the DNA level. Given that split inteins assemble essential proteins (e.g., DNA polymerase alpha subunit, DNA helicase, and ribonucleotide reductase), it is not surprising that this solution entails a number of traits that ensure highly efficient binding. Ultimately, the molecular details of this mechanism, laid out in this study, not only provide insight into the evolution of efficient biomolecular recognition, but they will also lay the groundwork for future engineering efforts on this unusual and useful class of proteins.
Experimental Section
ARTICLE SECTIONS
Sample Preparation
NpuN and NpuC were purified as previously described.(22) Isolated NpuN1 and NpuN2 proteins and the permuted intein constructs were generated similarly to NpuN. For functional analyses, wild-type sequences were used, and for biophysical measurements, a C1A mutation in NpuN and N137A mutation in NpuC were introduced to inactivate the intein. For segmental labeling of NpuN, the protein was split between Y58 and C59 and the two reactive fragments were produced and ligated using standard protocols. For isotope labeling, Escherichia coli were grown in minimal M9 medium enriched with 15NH4Cl and [U–13C]-d-glucose as the sole sources of nitrogen and carbon.
Biophysical Measurements
SEC-MALS experiments were carried out on a Superdex 75 10/300 column (volume = 24 mL) at 4 °C. For MALS analysis, light scattering and refractive index measurements were averaged across the entire width above half of the peak height to extract the molar mass. All NMR experiments were carried out at 25 °C in a pH 6.5 phosphate buffer using standard techniques. For ANS fluorescence, samples were excited at 370 nm and emission spectra were recorded between 400 and 720 nm. For steady-state tryptophan fluorescence, samples were excited at 290 or 295 nm and emission was recorded between 300 and 500 nm. For stopped-flow tryptophan fluorescence, samples were excited at 290 nm and emission was recorded above a 320 nm cutoff filter.
ARTICLE SECTIONS
Full methods and experimental data, including protein semisynthesis and purification protocols, characterization of proteins, and additional biophysical data. This material is available free of charge via the Internet at http://pubs.acs.org.
§Author Contributions
N.H.S. and E.E. contributed equally to this work.
The authors declare no competing financial interest.
Terms & Conditions
Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
ARTICLE SECTIONS
The authors thank the members of the Muir laboratory for valuable discussions and Istvan Pelczer of the Princeton University NMR facility for his generosity. This work was supported by the U.S. National Institutes of Health (NIH grant GM086868). NMR resources at NYSBC were supported by NIGMS P41-GM066354.
References
ARTICLE SECTIONS
This article references 30 other publications.
- 1Saleh, L.; Perler, F. B. Chem. Rec. 2006, 6, 183– 93[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XptlymsLs%253D&md5=adc1e1637c25e42f418d240e1cf080e2Protein splicing In Cis and In TransSaleh, Lana; Perler, Francine B.Chemical Record (2006), 6 (4), 183-193CODEN: CRHEAK; ISSN:1527-8999. (John Wiley & Sons, Inc.)A review. Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallog. study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were detd. using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.
- 2Pietrokovski, S. Trends Genet. 2001, 17, 465– 72[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlsFylu7s%253D&md5=149124813929befcde02e4d67d8f8887Intein spread and extinction in evolutionPietrokovski, S.Trends in Genetics (2001), 17 (8), 465-472CODEN: TRGEE2; ISSN:0168-9525. (Elsevier Science Ltd.)A review. Inteins are selfish DNA elements found within coding regions. They are translated with their host protein, but then catalyze their own excision and the formation of a peptide bond between their flanking protein regions. Understanding what drives and selects inteins is relevant for assessing whether they have unidentified biol. functions and whether they can invade and become established in new genes and organisms. Inteins are suggested to have been present and more common in the progenitors of eukaryotes and prokaryotes. In these cells, inteins had some beneficial function or had evolved from an unknown beneficial protein. Since then, this putative benefit has been lost and inteins are gradually becoming extinct. The proteins in which inteins are currently found are proposed to be proteins vital for the survival of the organism, where intein removal is most difficult.
- 3Perler, F. B. Nucleic Acids Res. 2002, 30, 383– 4[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xht12rsb0%253D&md5=fa2463b3bd0fbdd25f28cba1fba0fe51InBase: the Intein DatabasePerler, Francine B.Nucleic Acids Research (2002), 30 (1), 383-384CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliog. An intein-specific BLAST server is now available to assist in identifying new inteins.
- 4Vila-Perelló, M.; Muir, T. W. Cell 2010, 143, 191– 200[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht12qurnM&md5=12f45692e4a8d7b87e72799d03a300abBiological Applications of Protein SplicingVila-Perello, Miquel; Muir, Tom W.Cell (Cambridge, MA, United States) (2010), 143 (2), 191-200CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a mol. disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chem. and biol., allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.
- 5Caspi, J.; Amitai, G.; Belenkiy, O.; Pietrokovski, S. Mol. Microbiol. 2003, 50, 1569– 77[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVShtb3I&md5=6f713ce6753313b31ff104f4fc6f4459Distribution of split DnaE inteins in cyanobacteriaCaspi, Jonathan; Amitai, Gil; Belenkiy, Olga; Pietrokovski, ShmuelMolecular Microbiology (2003), 50 (5), 1569-1577CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analyzing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnol.
- 6Dassa, B.; London, N.; Stoddard, B. L.; Schueler-Furman, O.; Pietrokovski, S. Nucleic Acids Res. 2009, 37, 2560– 73[Crossref], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlsleru7c%253D&md5=47ca9d9d02a3be15d575b019473e0e5aFractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease familyDassa, Bareket; London, Nir; Stoddard, Barry L.; Schueler-Furman, Ora; Pietrokovski, ShmuelNucleic Acids Research (2009), 37 (8), 2560-2573CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic anal. of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this fractured' organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) endonucleases, was found in half of the loci. These putative homing endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.
- 7Southworth, M. W.; Adam, E.; Panne, D.; Byer, R.; Kautz, R.; Perler, F. B. EMBO J. 1998, 17, 918– 26[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhs1yrtLk%253D&md5=be87a8099c13aff8d8ce7d8ddd3a2120Control of protein splicing by intein fragment reassemblySouthworth, Maurice W.; Adam, Eric; Panne, Daniel; Byer, Robyn; Kautz, Roger; Perler, Francine B.EMBO Journal (1998), 17 (4), 918-926CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in sep. hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endo-nuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or contg. affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not obsd. with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR anal. or enabling the study of the effects of any type of protein modification on a limited region of a protein.
- 8Yamazaki, T.; Otomo, T.; Oda, N.; Kyogoku, Y.; Uegaki, K.; Ito, N.; Ishino, Y.; Nakamura, H. J. Am. Chem. Soc. 1998, 120, 5591– 5592[ACS Full Text
], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtlWku7o%253D&md5=df604b078a8bcd7ffc9d4293a151b694Segmental Isotope Labeling for Protein NMR Using Peptide SplicingYamazaki, Toshio; Otomo, Takanori; Oda, Natsuko; Kyogoku, Yoshimasa; Uegaki, Koichi; Ito, Nobutoshi; Ishino, Yoshizumi; Nakamura, HarukiJournal of the American Chemical Society (1998), 120 (22), 5591-5592CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)This article demonstrates a new method for the ligation of labeled and unlabeled peptide fragments using a peptide splicing element, intein (Protozyme). This method is expected to facilitate the application of NMR to proteins as large as 50 kDa. Segmental labeling will make it possible to assign signals unambiguously in a short time and to det. the structures of such large proteins to a higher precision. - 9Mootz, H. D.; Blum, E. S.; Tyszkiewicz, A. B.; Muir, T. W. J. Am. Chem. Soc. 2003, 125, 10561– 9[ACS Full Text], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmtVKntLs%253D&md5=6626d6a79010d4cfa48a1f4e437c216cConditional protein splicing: A new tool to control protein structure and function in vitro and in vivoMootz, Henning D.; Blum, Elyse S.; Tyszkiewicz, Amy B.; Muir, Tom W.Journal of the American Chemical Society (2003), 125 (35), 10561-10569CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small mol. rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunopptn. in cells treated with rapamycin or a nontoxic analog thereof. No background splicing in the absence of the small-mol. inducer was obsd. over a 24-h time course. Product formation could be detected within 10 min of addn. of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small mol. ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.
- 10Oeemig, J. S.; Aranko, A. S.; Djupsjöbacka, J.; Heinämäki, K.; Iwaï, H. FEBS Lett. 2009, 583, 1451– 1456[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlsVKgsbw%253D&md5=1985910fe023b377877ccca6c61e175cSolution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site-specific chemical modificationOeemig, Jesper S.; Aranko, A. Sesilja; Djupsjoebacka, Janica; Heinaemaeki, Kimmo; Iwai, HideoFEBS Letters (2009), 583 (9), 1451-1456CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We detd. the soln. structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.
- 11Shi, J.; Muir, T. W. J. Am. Chem. Soc. 2005, 127, 6198– 206[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjtVWntr4%253D&md5=f2eb1818bd9e802e55faace23424dbecDevelopment of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split InteinsShi, Jianxin; Muir, Tom W.Journal of the American Chemical Society (2005), 127 (17), 6198-6206CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chem. biol. and biotechnol., precious little is known about the mol. recognition events governing the initial fragment assocn. step. In this study, fluorescence approaches have been used to measure the dissocn. const. for the Ssp DnaE split intein interaction and to det. the on and off rates of fragment assocn. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid assocn. of the fragments at physiol. pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technol. should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins.
- 12Shah, N. H.; Vila-Perelló, M.; Muir, T. W. Angew. Chem., Int. Ed. 2011, 50, 6511– 5[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnt1eitbg%253D&md5=98573036657447152bc6a05d5a37adb9Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split inteinShah, Neel H.; Vila-Perello, Miquel; Muir, Tom W.Angewandte Chemie, International Edition (2011), 50 (29), 6511-6515, S6511/1-S6511/45CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)While ionic interactions have previously been postulated to play a role in split intein assembly, the involvement of electrostatic forces has not been validated exptl. The authors set out to test the hypothesis that ionic interactions facilitate the assocn. of split intein fragments and thus could be manipulated to control the relative reactivities of different N- and C-intein complexes. This study probed the role of intermol. ion clusters for fragment assembly and splicing in the NpuWT split intein both in vivo and in vitro. Through these expts., the authors rationally designed a new split intein, NpuMUT, which displays low cross-reactivity with NpuWT. These orthogonal inteins were used to generate the large, full-length, active mammalian protein PARP1 through a one-pot three-piece ligation. Collectively, the results demonstrate that electrostatic interactions can engender kinetic control in a complex enzymic system. Furthermore, these results provide insight into the mol. requirements for efficient and specific protein trans-splicing.
- 13Zheng, Y.; Wu, Q.; Wang, C.; Xu, M.-Q.; Liu, Y. Biosci. Rep. 2012, 32, 433– 42[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlCjsb3N&md5=86818bead0373351bc14a6416f4fe33cMutual synergistic protein folding in split inteinZheng, Yuchuan; Wu, Qin; Wang, Chunyu; Xu, Min-qun; Liu, YangzhongBioscience Reports (2012), 32 (5), 433-442CODEN: BRPTDT; ISSN:0144-8463. (Portland Press Ltd.)Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two sepd. halves, and the recognition and assocn. of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodn. anal. on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon assocn. ITC (isothermal titrn. calorimetry) reveals that the highly favorable enthalpy change drives the assocn. of the two halves, overcoming the unfavorable entropy change. The high flexibility of two fragments and the marked thermodn. preference provide a robust assocn. for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein.
- 14Zettler, J.; Schütz, V.; Mootz, H. D. FEBS Lett. 2009, 583, 909– 14[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXis1aqtb0%253D&md5=6ee44ff008bd1d4d44f73c960671b716The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reactionZettler, Joachim; Schuetz, Vivien; Mootz, Henning D.FEBS Letters (2009), 583 (5), 909-914CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)We have studied the naturally split α subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and detd. an apparent first-order rate const. of (1.1 ± 0.2) × 10-2 s-1 at 37 °C. This represents the highest rate reported to date for the protein trans-splicing reaction (t1/2 of ∼60 s). Furthermore, the reaction was very robust and exhibited high yield with respect to extein sequence, temp. (6 to 37°C), and urea (up to 6 M). Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chem.
- 15Shah, N. H.; Dann, G. P.; Vila-Perelló, M.; Liu, Z.; Muir, T. W. J. Am. Chem. Soc. 2012, 134, 11338– 41[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptF2iu7Y%253D&md5=dd6b4d96222fde82854c206d01128851Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein EngineeringShah, Neel H.; Dann, Geoffrey P.; Vila-Perello, Miquel; Liu, Zhihua; Muir, Tom W.Journal of the American Chemical Society (2012), 134 (28), 11338-11341CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly obsd. for this autoprocessing protein family. Furthermore, when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chem. biol.
- 16Muona, M.; Aranko, A. S.; Raulinaitis, V.; Iwaï, H. Nat. Protoc. 2010, 5, 574– 87[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislOmsLs%253D&md5=b5dbc0b472c9ae567a8ebcd79bcb5ac1Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitroMuona, Mikko; Aranko, A. Sesilja; Raulinaitis, Vytas; Iwai, HideoNature Protocols (2010), 5 (3), 574-587CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Segmental isotopic labeling is a powerful labeling technique for reducing NMR signal overlap, which is assocd. with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR expts. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or α-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total prepn. time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.
- 17Borra, R.; Dong, D.; Elnagar, A. Y.; Woldemariam, G. A.; Camarero, J. A. J. Am. Chem. Soc. 2012, 134, 6344– 53[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1WksrY%253D&md5=578e1f4afc767f2f0844dd66320e5017In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split InteinsBorra, Radhika; Dong, Dezheng; Elnagar, Ahmed Y.; Woldemariam, Getachew A.; Camarero, Julio A.Journal of the American Chemical Society (2012), 134 (14), 6344-6353CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Methods to visualize, track, and modify proteins in living cells are central for understanding the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chem. labeling of proteins. The authors report in this work the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. The authors have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, the authors have shown that this approach can be also used for modifying proteins to control their cellular localization and potentially alter their biol. activity.
- 18Vila-Perelló, M.; Liu, Z.; Shah, N. H.; Willis, J. A.; Idoyaga, J.; Muir, T. W. J. Am. Chem. Soc. 2013, 135, 286– 92[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvV2gsL3E&md5=ea910b6e58fc18fa6f5da446d117af1aStreamlined Expressed Protein Ligation Using Split InteinsVila-Perello, Miquel; Liu, Zhihua; Shah, Neel H.; Willis, John A.; Idoyaga, Juliana; Muir, Tom W.Journal of the American Chemical Society (2013), 135 (1), 286-292CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. modified proteins are invaluable tools for studying the mol. details of biol. processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-contg. peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. The authors describe here a new method for the prepn. and purifn. of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to assoc. to become active. Taking advantage of the strong affinity between the two split intein fragments, the authors devised a streamlined procedure for the purifn. and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.
- 19Jagadish, K.; Borra, R.; Lacey, V.; Majumder, S.; Shekhtman, A.; Wang, L.; Camarero, J. A. Angew. Chem., Int. Ed. 2013, 52, 3126– 31[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXos1agug%253D%253D&md5=f32c6c42f1a3c7783a8c737117dd07f4Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide-Protein InteractionsJagadish, Krishnappa; Borra, Radikha; Lacey, Vanessa; Majumder, Subhabrata; Shekhtman, Alexander; Wang, Lei; Camarero, Julio A.Angewandte Chemie, International Edition (2013), 52 (11), 3126-3131CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors showed that the biosynthesis of cyclotides contg. non-natural amino acids can be achieved by using different intein-based methods. EPL-backbone cyclization can provide non-natural amino acid contg. cyclotides when the cyclization is carried out in vitro by GSH-induced cyclization and folding of the corresponding precursor. In vivo prodn., however, is less efficient using this method. It was shown that PTS-mediated backbone cyclization using the highly efficient Npu DnaE split-intein can be used for the efficient prodn. of cyclotides inside live E. coli cells. It was estd. that the in vivo prodn. of MCoTI-I was around seven times more efficient using Npu DnaE PTS than EPL, thereby providing an attractive alternative for the prodn. of these types of polypeptides in bacterial cells. The high efficiency of PTS-mediated cyclization combined with nonsense-suppressing orthogonal tRNA/synthetase technol. made the in vivo prodn. of cyclotides contg. non-natural amino acids possible. Of particular interest is the introduction of azido-contg. amino acids, which can react with DBCO-contg. fluorescent probes to provide in vivo fluorescently labeled cyclotides. Cyclotides contg. the nonnatural amino acid AziF can be expressed in live bacterial cells and easily labeled with DBCO-AMCA to monitor cyclotide-protein interactions. This finding opens the possibility for in vitro and potentially also in vivo screening of genetically encoded libraries of cyclotides for the rapid selection of novel cyclotide sequences able to bind a specific bait protein using high-throughput cell-based optical screening approaches.
- 20Sorci, M.; Dassa, B.; Liu, H.; Anand, G.; Dutta, A. K.; Pietrokovski, S.; Belfort, M.; Belfort, G. Anal. Chem. 2013, 85, 6080– 8[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvVCqtrk%253D&md5=5a745992f179e62da10f2310600d7c0eOriented Covalent Immobilization of Antibodies for Measurement of Intermolecular Binding Forces between Zipper-Like Contact Surfaces of Split InteinsSorci, Mirco; Dassa, Bareket; Liu, Hongwei; Anand, Gaurav; Dutta, Amit K.; Pietrokovski, Shmuel; Belfort, Marlene; Belfort, GeorgesAnalytical Chemistry (Washington, DC, United States) (2013), 85 (12), 6080-6088CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In order to measure the intermol. binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermol. force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (∼52 pN). Bioinformatic sequence anal. and computational structural anal. discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, the authors tested the Jarzynski's equality and demonstrated, as expected, that nonequil. dissipative measurements obtained here gave larger energies of interaction as compared with those for equil. Hence, AFM coupled with the authors' immobilization strategy and computational studies provides a useful anal. tool for the direct measurement of intermol. assocn. of split inteins and could be extended to any interacting protein pair.
- 21Shah, N. H.; Muir, T. W. Isr. J. Chem. 2011, 51, 854– 861[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2gsbzP&md5=c51573533e69199e56a4de578d92981fSplit inteins: Nature's protein ligasesShah, Neel H.; Muir, Tom W.Israel Journal of Chemistry (2011), 51 (8-9), 854-861CODEN: ISJCAT; ISSN:0021-2148. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Split inteins carry out a naturally occurring process known as protein trans-splicing, where 2 protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past 13 yr since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a no. of biotechnol. applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, the authors discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.
- 22Shah, N. H.; Eryilmaz, E.; Cowburn, D.; Muir, T. W. J. Am. Chem. Soc. 2013, 135, 5839– 47[ACS Full Text], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktFKgtLg%253D&md5=0b249056e530bcd593e886755ab66332Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-SplicingShah, Neel H.; Eryilmaz, Ertan; Cowburn, David; Muir, Tom W.Journal of the American Chemical Society (2013), 135 (15), 5839-5847CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Split inteins play an important role in modern protein semi-synthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic redn. in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here a thorough kinetic anal. of the relationship between C-extein compn. and split intein activity is presented. The results of these expts. were used to guide structural and mol. dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, the structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.
- 23Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Proteins: Struct., Funct., Genet. 2000, 41, 415– 427[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnslGnsLs%253D&md5=ddd4a8ba1d035c3d566e1a50b509a173Why are "Natively unfolded" proteins unstructured under physiologic conditions?Uversky, Vladimir N.; Gillespie, Joel R.; Fink, Anthony L.Proteins: Structure, Function, and Genetics (2000), 41 (3), 415-427CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)"Natively unfolded" proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Anal. of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and "natively unfolded" ones. The results show that "natively unfolded" proteins are specifically localized within a unique region of charge-hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of "natively unfolded" proteins.
- 24Müller-Späth, S.; Soranno, A.; Hirschfeld, V.; Hofmann, H.; Rüegger, S.; Reymond, L.; Nettels, D.; Schuler, B. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14609– 14[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVOgsb7K&md5=8b1083dd092c95fba89c18bf4b70394dCharge interactions can dominate the dimensions of intrinsically disordered proteinsMuller-Spath, Sonja; Soranno, Andrea; Hirschfeld, Verena; Hofmann, Hagen; Ruegger, Stefan; Reymond, Luc; Nettels, Daniel; Schuler, BenjaminProceedings of the National Academy of Sciences of the United States of America (2010), 107 (33), 14609-14614, S14609/1-S14609/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many eukaryotic proteins are disordered under physiol. conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-mol. Forster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been obsd. for many proteins at low denaturant concn., IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an addnl. collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.
- 25Semisotnov, G. V.; Rodionova, N. A.; Razgulyaev, O. I.; Uversky, V. N.; Gripas, A. F.; Gilmanshin, R. I. Biopolymers 1991, 31, 119– 128[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhvVWmurY%253D&md5=e0d4a2d45d8d8482cfdc1d21b15eebfeStudy of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probeSemisotnov, G. V.; Rodionova, N. A.; Razgulyaev, O. I.; Uverskii, V. N.; Gripas, A. F.; Gil'manshin, R. I.Biopolymers (1991), 31 (1), 119-28CODEN: BIPMAA; ISSN:0006-3525.Binding of the hydrophobic fluorescent probe 1-anilino-naphthalene-8-sulfonate (ANS) to synthetic polypeptides and proteins with a different structural organization was studied. It was shown that ANS has a much stronger affinity to the protein molten globule state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, α-helical, or β-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equil. and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, β-lactamase, and α-lactalbumin as examples.
- 26Xu, R.; Ayers, B.; Cowburn, D.; Muir, T. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 388– 93[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmtlCgsg%253D%253D&md5=b44cd6f792c3957b0c5b7dfc59807b2fChemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studiesXu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (2), 388-393CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A convenient in vitro chem. ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homol. type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory app. of the protein, were individually prepd. in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prep. NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prep. recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chem. ligation in vitro, will increase the size limits for protein structural detn. in soln. with NMR methods. In vitro chem. ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
- 27Millet, O.; Loria, J. P.; Kroenke, C. D.; Pons, M.; Palmer, A. G. J. Am. Chem. Soc. 2000, 122, 2867– 2887[ACS Full Text], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Crsbs%253D&md5=d2e62cf97ded2433edaebae5b7ffe704The Static Magnetic Field Dependence of Chemical Exchange Linebroadening Defines the NMR Chemical Shift Time ScaleMillet, Oscar; Loria, J. Patrick; Kroenke, Christopher D.; Pons, Miquel; Palmer, Arthur G., IIIJournal of the American Chemical Society (2000), 122 (12), 2867-2877CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The static magnetic field dependence of chem. exchange linebroadening in NMR spectroscopy is investigated theor. and exptl. Two-site exchange (A ↹ B) is considered with site A more highly populated than site B (pa > pb), a shift difference between sites equal to Δω, and an exchange rate const. given by kex. The exchange contribution to the transverse relaxation rate const. for the more highly populated site is denoted Rex. The dependence of Rex on the static magnetic field strength is characterized by a scaling parameter α = d ln Rex/d ln Δω, in which 0 ≤ α ≤ 2 for pa > 0.7. The value of α depends on the NMR chem. shift time scale for the exchange process: for slow exchange (kex/Δω < 1), 0 ≤ α < 1; for intermediate exchange (kex/Δω = 1), α = 1; and for fast exchange (kex/Δω > 1), 1 < α ≤ 2. Consequently, the static magnetic field dependence of Rex defines the chem. shift time scale for an exchange process even if the populations are so highly skewed (pa » pb) that the minor resonance is not observable in the slow exchange limit. The theor. results are verified by measuring 15N transverse relaxation rate consts. at static magnetic fields of 11.7 and 14.1 T and temps. of 300 and 313 K for the protein basic pancreatic trypsin inhibitor. At each combination of static magnetic field and temp., the rate consts. were measured using Carr-Purcell-Meiboom-Gill and Hahn echo techniques with spin-echo delays ranging from 1.0 to 64.5 ms. 15N resonances for residues in the region of the Cys14-Cys38 disulfide bond are broadened due to chem. exchange. Values of α obtained from the relaxation rate consts. range from 0.26 ± 0.17 for Arg39 at 300 K to 1.96 ± 0.25 for Cys38 at 313 K. For Cys38 and Arg39, the two residues most strongly affected by chem. exchange, values of kex were detd. to be 380 ± 70 s-1 and 530 ± 90 s-1 at 300 K and 1300 ± 290 s-1 and 1370 ± 160 s-1 at 313 K by global anal. of the relaxation rate consts. The scaling parameters α indicate that chem. exchange for most residues in basic pancreatic trypsin inhibitor does not satisfy kex/Δω » 1. Consequently, the assumption of fast-limit quadratic scaling of exchange broadening in proteins and other macromols. may be incorrect, even if a single broadened resonance is obsd. for a nuclear spin. The theor. results for the static magnetic field dependence of chem. exchange broadening in NMR spectroscopy are applicable to other nuclei and to other techniques for measuring chem. exchange linebroadening.
- 28Shoemaker, B. A.; Portman, J. J.; Wolynes, P. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8868– 73[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXls12ltLY%253D&md5=614acf652b32372b4d90f53fb64070a8Speeding molecular recognition by using the folding funnel: the fly-casting mechanismShoemaker, Benjamin A.; Portman, John J.; Wolynes, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (16), 8868-8873CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein mol. can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the "fly-casting mechanism.". We illustrate this scenario with the hypothetical kinetics of binding a single repressor mol. to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.
- 29Schreiber, G.; Fersht, A. R. Nat. Struct. Biol. 1996, 3, 427– 31[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XivVahtrk%253D&md5=f2b4e71b355b469c425d055f4d9562f3Rapid, electrostatically assisted association of proteinsSchreiber, Gideon; Fersht, Alan R.Nature Structural Biology (1996), 3 (5), 427-431CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)The rapid assocn. of barnase and its intracellular inhibitor barstar has been analyzed from the effects of mutagenesis and electrostatic screening. A basal assocn. rate const. of 105 M-1 s-1 is increased to over 5 × 109 M-1 s-1 by electrostatic forces. The assocn. between the oppositely charged proteins proceeds through the rate-detg. formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase assocn. rate consts. between mols. and its principles may be used for protein design.
- 30Choi, J. J.; Nam, K. H.; Min, B.; Kim, S.-J.; Söll, D.; Kwon, S.-T. J. Mol. Biol. 2006, 356, 1093– 106[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlGmtr8%253D&md5=c62f01ce7e79634f7c219705ab343487Protein Trans-splicing and Characterization of a Split Family B-type DNA Polymerase from the Hyperthermophilic Archaeal Parasite Nanoarchaeum equitansChoi, Jeong Jin; Nam, Ki Hoon; Min, Bokkee; Kim, Sang-Jin; Soell, Dieter; Kwon, Suk-TaeJournal of Molecular Biology (2006), 356 (5), 1093-1106CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two sep. genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80° after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examd. in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temp., and occurred only at temps. above 50°. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'→5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'→5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show exptl. that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.
Cited By
This article is cited by 36 publications.
- Robert E. Thompson, Tom W. Muir. Chemoenzymatic Semisynthesis of Proteins. Chemical Reviews 2019, Article ASAP.
- Josef A. Gramespacher, Antony J. Burton, Luis F. Guerra, Tom W. Muir. Proximity Induced Splicing Utilizing Caged Split Inteins. Journal of the American Chemical Society 2019, 141 (35) , 13708-13712. DOI: 10.1021/jacs.9b05721.
- Eugene Kim, Bin Dai, James B. Qiao, Wenlu Li, John D. Fortner, Fuzhong Zhang. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Applied Materials & Interfaces 2018, 10 (49) , 43003-43012. DOI: 10.1021/acsami.8b14890.
- Adam J. Stevens, Giridhar Sekar, Josef A. Gramespacher, David Cowburn, Tom W. Muir. An Atypical Mechanism of Split Intein Molecular Recognition and Folding. Journal of the American Chemical Society 2018, 140 (37) , 11791-11799. DOI: 10.1021/jacs.8b07334.
- Gongyu Li, Siming Yuan, Shihui Zheng, Yangzhong Liu, Guangming Huang. In Situ Living Cell Protein Analysis by Single-Step Mass Spectrometry. Analytical Chemistry 2018, 90 (5) , 3409-3415. DOI: 10.1021/acs.analchem.7b05055.
- Josef A. Gramespacher, Adam J. Stevens, Duy P. Nguyen, Jason W. Chin, and Tom W. Muir . Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. Journal of the American Chemical Society 2017, 139 (24) , 8074-8077. DOI: 10.1021/jacs.7b02618.
- Adam J. Stevens, Zachary Z. Brown, Neel H. Shah, Giridhar Sekar, David Cowburn, and Tom W. Muir . Design of a Split Intein with Exceptional Protein Splicing Activity. Journal of the American Chemical Society 2016, 138 (7) , 2162-2165. DOI: 10.1021/jacs.5b13528.
- Jun Liu, Oshini Ekanayake, Dominic Santoleri, Kelsi Walker, Sharon Rozovsky. Efficient Generation of Hydrazides in Proteins by RadA Split Intein. ChemBioChem 2020, 21 (3) , 346-352. DOI: 10.1002/cbic.201900160.
- Georgeta N Basturea. Inteins. Materials and Methods 2020, 10 DOI: 10.13070/mm.en.10.2868.
- Hannes M. Beyer, Kornelia M. Mikula, Mi Li, Alexander Wlodawer, Hideo Iwaï. The crystal structure of the naturally split gp41‐1 intein guides the engineering of orthogonal split inteins from cis ‐splicing inteins. The FEBS Journal 2019, 265 DOI: 10.1111/febs.15113.
- Hannes M. Beyer, Hideo Iwaï. Off‐Pathway‐Sensitive Protein‐Splicing Screening Based on a Toxin/Antitoxin System. ChemBioChem 2019, 20 (15) , 1933-1938. DOI: 10.1002/cbic.201900139.
- Kevin W Keating, Eric M Young. Synthetic biology for bio-derived structural materials. Current Opinion in Chemical Engineering 2019, 24, 107-114. DOI: 10.1016/j.coche.2019.03.002.
- Andrzej Stanisław Cieplak. Tau Inclusions in Alzheimer's, Chronic Traumatic Encephalopathy and Pick's Disease. A Speculation on How Differences in Backbone Polarization Underlie Divergent Pathways of Tau Aggregation. Frontiers in Neuroscience 2019, 13 DOI: 10.3389/fnins.2019.00488.
- Barbara Di Ventura, Henning D. Mootz. Switchable inteins for conditional protein splicing. Biological Chemistry 2019, 400 (4) , 467-475. DOI: 10.1515/hsz-2018-0309.
- Shubhendu Palei, Kira S. Becher, Christian Nienberg, Joachim Jose, Henning D. Mootz. Bacterial Cell‐Surface Display of Semisynthetic Cyclic Peptides. ChemBioChem 2019, 20 (1) , 72-77. DOI: 10.1002/cbic.201800552.
- Yi-Zong Lee, Shih-Che Sue. Salt-sensitive intein for large-scale polypeptide production. 2019,,, 111-130. DOI: 10.1016/bs.mie.2019.02.024.
- Dominique Richardson, Jaakko Itkonen, Julia Nievas, Arto Urtti, Marco G. Casteleijn. Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation. Scientific Reports 2018, 8 (1) DOI: 10.1038/s41598-018-30435-4.
- Yanran Xu, Lei Zhang, Buyong Ma, Lifu Hu, Huili Lu, Tonglu Dou, Junsheng Chen, Jianwei Zhu. Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE. Enzyme and Microbial Technology 2018, 118, 6-12. DOI: 10.1016/j.enzmictec.2018.06.013.
- Verónica Gordo, David Aparicio, Rosa Pérez-Luque, Antoni Benito, Maria Vilanova, Isabel Usón, Ignacio Fita, Marc Ribó. Structural Insights into Subunits Assembly and the Oxyester Splicing Mechanism of Neq pol Split Intein. Cell Chemical Biology 2018, 25 (7) , 871-879.e2. DOI: 10.1016/j.chembiol.2018.04.008.
- Josef A. Gramespacher, Adam J. Stevens, Robert E. Thompson, Tom W. Muir. Improved protein splicing using embedded split inteins. Protein Science 2018, 27 (3) , 614-619. DOI: 10.1002/pro.3357.
- Marco G. Casteleijn, Dominique Richardson, Petteri Parkkila, Niko Granqvist, Arto Urtti, Tapani Viitala. Spin coated chitin films for biosensors and its analysis are dependent on chitin-surface interactions. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2018, 539, 261-272. DOI: 10.1016/j.colsurfa.2017.12.036.
- Merideth A. Cooper, Joseph E. Taris, Changhua Shi, David W. Wood. A Convenient Split‐Intein Tag Method for the Purification of Tagless Target Proteins. Current Protocols in Protein Science 2018, 91 (1) DOI: 10.1002/cpps.46.
- Leonhard M. Kick, Sabrina Harteis, Maximilian F. Koch, Sabine Schneider. Mechanistic Insights into Cyclic Peptide Generation by DnaE Split-Inteins through Quantitative and Structural Investigation. ChemBioChem 2017, 18 (22) , 2242-2246. DOI: 10.1002/cbic.201700503.
- Alexander Muik, Johanna Reul, Thorsten Friedel, Anke Muth, Karen Patricia Hartmann, Irene C. Schneider, Robert C. Münch, Christian J. Buchholz. Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer. Biomaterials 2017, 144, 84-94. DOI: 10.1016/j.biomaterials.2017.07.032.
- Andrzej Stanisław Cieplak, . Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions. PLOS ONE 2017, 12 (9) , e0180905. DOI: 10.1371/journal.pone.0180905.
- Tobias Schubeis, Madhu Nagaraj, Christiane Ritter. Segmental Isotope Labeling of Insoluble Proteins for Solid-State NMR by Protein Trans-Splicing. 2017,,, 147-160. DOI: 10.1007/978-1-4939-6451-2_10.
- Galia T. Debelouchina, Tom W. Muir. A molecular engineering toolbox for the structural biologist. Quarterly Reviews of Biophysics 2017, 50 DOI: 10.1017/S0033583517000051.
- Chelsea M. Stewart, Cosmo Z. Buffalo, J. Andrés Valderrama, Anna Henningham, Jason N. Cole, Victor Nizet, Partho Ghosh. Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction. Proceedings of the National Academy of Sciences 2016, 113 (34) , 9515-9520. DOI: 10.1073/pnas.1606160113.
- Anne-Lena Bachmann, Julian C. J. Matern, Vivien Schütz, Henning D. Mootz. Chemical-Tag Labeling of Proteins Using Fully Recombinant Split Inteins. 2015,,, 145-159. DOI: 10.1007/978-1-4939-2272-7_10.
- Jerome M. Karp, Ertan Erylimaz, David Cowburn. Correlation of chemical shifts predicted by molecular dynamics simulations for partially disordered proteins. Journal of Biomolecular NMR 2015, 61 (1) , 35-45. DOI: 10.1007/s10858-014-9879-2.
- Shelly DeForte, Krishna D Reddy, Vladimir N Uversky. Digested disorder, Quarterly intrinsic disorder digest (October-November-December, 2013). Intrinsically Disordered Proteins 2015, 3 (1) , e984569. DOI: 10.4161/21690707.2014.984569.
- Bastian Groitl, Ursula Jakob. Thiol-based redox switches. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2014, 1844 (8) , 1335-1343. DOI: 10.1016/j.bbapap.2014.03.007.
- Yi-Zong Lee, Yun-Tzai Lee, Yi-Jan Lin, Yen-Ju Chen, Shih-Che Sue. A streamlined method for preparing split intein for NMR study. Protein Expression and Purification 2014, 99, 106-112. DOI: 10.1016/j.pep.2014.04.005.
- Ertan Eryilmaz, Neel H. Shah, Tom W. Muir, David Cowburn. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. Journal of Biological Chemistry 2014, 289 (21) , 14506-14511. DOI: 10.1074/jbc.R113.540302.
- Zhipeng Wang, Xiaozhe Ding, Sijian Li, Jing Shi, Yiming Li. Engineered fluorescence tags for in vivo protein labelling. RSC Adv. 2014, 4 (14) , 7235-7245. DOI: 10.1039/C3RA46991C.
- Neel H. Shah, Tom W. Muir. Inteins: nature's gift to protein chemists. Chem. Sci. 2014, 5 (2) , 446-461. DOI: 10.1039/C3SC52951G.
Abstract
Figure 1
Figure 1. Characterization of Npu fragments and complex. (a) Scheme depicting the genetic origin of split intein fragments and their role in protein trans-splicing. (b) Rendering of the Npu intein structure, highlighting relevant NpuN lobes (NpuN1 in dark blue and NpuN2 in light blue) and NpuC in red. Termini to which exteins would be attached are shown as orange spheres, and sites of intrinsic fluorophores used in this study (W47 and Y58W) are shown as green sticks; rendering based on PDB 2KEQ.(10) (c) SEC-MALS of NpuN (blue, 25 μM, expected MW = 12188 Da), NpuC (red, 400 μM, expected MW = 4443 Da), and their complex (black, 25 μM, expected MW = 16631 Da). (d) 1H–15N HSQC spectra of NpuC alone (red, 250 μM, 600 MHz) and in complex with unlabeled NpuN (black, 250 μM, 800 MHz). (e) 1H–15N HSQC spectra of NpuN alone (blue, 250 μM, 500 MHz) and in complex with unlabeled NpuC (black, 250 μM, 800 MHz). NMR spectra were acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
Figure 2
Figure 2. Electrostatic surface representations of Npu. The color scheme for these representations is red for negative charge, white for neutral, and blue for positive charge. All perspectives are given relative to panel (a), which is the same perspective as Figure 1b. The renderings are based on PDB 2KEQ.(10) Panels a and b highlight complementary hydrophobic surfaces on either fragment at the NpuN1–NpuC interface. Panels c and d highlight complementary electrostatic surfaces on either fragment at the NpuN2–NpuC interface.
Figure 3
Figure 3. Characterization of the NpuN lobes. (a) Charge-hydrophobicity plot comparing Npu fragments, complex, and N-intein lobes. Mean hydrophobicity (H) is calculated on a normalized Kyte-Doolittle scale, and mean net charge (R) is the absolute value. The solid line delineating disordered and folded proteins is empirically defined as R = 2.785·H – 1.151.(23) (b) SEC of NpuN1 (dark blue, 7 μM), NpuN2 (light blue, 7 μM) and NpuC (red, 7 μM). (c) Scheme depicting the segmental isotopic labeling of NpuN using Expressed Protein Ligation. (d) 1H–15N HSQC spectra of NpuN segmentally 15N-labeled on NpuN1 (dark blue, 100 μM) and NpuN2 (light blue, 100 μM) collected at 500 MHz. NMR data was acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
Figure 4
Figure 4. Characterization of the NpuN2–NpuC interaction. (a) SEC-MALS of isolated NpuN2 (light blue, 62.5 μM, expected MW = 6098 Da), NpuC (red, 7 μM), and an equimolar mixture of NpuN2 and NpuC (black, 50 μM, expected MW = 10541 Da). (b) 1H–15N HSQC spectra of NpuN2 alone (light blue, 250 μM) and with equimolar unlabeled NpuC (black, 250 μM) collected at 800 MHz. (c) Chemical shift index values (ΔC/H = δC/HObserved – δC/HRandomCoil) for Cα, CO, and HN atoms for NpuN2 in complex with NpuC. The consensus secondary structure prediction is shown below compared with NpuN2 in the context of the full Npu complex (as seen in PDB 2KEQ).(10) The NpuN2 sequence is given above, with important anionic residues in orange. (d) Composite 1H and 15N backbone chemical shift perturbation values for NpuC in complex with NpuN (black) or NpuN2 (light blue) calculated relative to isolated NpuC. Δδi = [(ΔδH,complex – ΔδH,alone)i2 + 0.11(ΔδN,complex – ΔδN,alone)i2 ]1/2 The NpuC sequence is given above, with important cationic residues in orange. Below the sequence, an asterisk marks unassigned residues for isolated NpuC (red), the complex with NpuN (black), or the complex with NpuN2 (light blue). (e) Rendering of the NpuN2–NpuC interaction, as seen in the native Npu complex (PDB 2KEQ).(10) NpuN2 is light blue, NpuC is red, and charged residues involved in intermolecular electrostatic interactions are highlighted as orange sticks. Predicted β-sheet and α-helical regions of NpuN2 are colored in green and purple, respectively. Residues 103–115 of NpuC are rendered as a ribbon, and the remainder of the sequence is rendered as a dotted line. (f) 1H–15N HSQC spectrum of Npu(51–115) collected at 600 MHz. NMR data in panels b, c, d, and f were acquired at 25 °C in a pH 6.5 buffer containing 25 mM phosphates, 100 mM NaCl, and 1 mM DTT.
Figure 5
Figure 5. Intrinsic fluorescence binding measurements: (a) Tryptophan fluorescence of wild-type NpuN in the absence and presence of NpuC (λex = 290 nm); (b) Tryptophan fluorescence of wild-type Npu upon stopped-flow mixing of fragments (λex = 290 nm, λem = >320 nm); (c) Residuals from fits to a one-phase (top) and two-phase (bottom) binding model for wild-type Npu; (d) Tryptophan fluorescence of NpuN with the W47Y and Y58W mutations in the absence and presence of NpuC (λex = 290 nm); (e) Tryptophan fluorescence of Npu W47Y,Y58W upon stopped-flow mixing of fragments (λex = 290 nm, λem = >320 nm); (f) Residuals from fits to a one-phase (top) and two-phase (bottom) binding model for Npu W47Y,Y58W.
Figure 6
Figure 6. The “capture and collapse” mechanism of split intein assembly. An N-terminal segment of the disordered C-intein is captured by the extended second lobe of the N-intein with compaction of that lobe into a native-like structure. This intermediate then collapses as the remainder of the C-intein docks into the preorganized first lobe of the N-intein.
Figure 7
Figure 7. Split intein assembly and function with engineered sequence topologies. (a) Structural representation of the two intein regions in the permuted Npu constructs (based on PDB 2KEQ).(10) The dashed line represents a diglycine linker introduced between residues 1–50 and 116–137. (b) Scheme showing protein splicing with the permuted Npu. Ub (ubiquitin) and SUMO are model N- and C-exteins. (c) Proteins splicing of permuted Npu at 10 and 100 μM fragment concentrations. Reaction mixtures when analyzed at indicated time-points by SDS-PAGE with coomassie staining. (d) SEC-MALS of the three-piece Npu complex (black, 7 μM of each fragment, expected MW = 16649 Da) compared with the individual components (NpuN1, dark blue; NpuN2, light blue; NpuC, red; each at 7 μM). (e) Scheme showing protein splicing with the three-piece Npu. (f) Splicing activity of the three-piece Npu at 5 μM fragment concentrations. Reaction mixtures where analyzed at indicated time-points by SDS-PAGE with coomassie staining. The asterisk indicates a side product, most likely SUMO due to premature N-extein cleavage.
References
ARTICLE SECTIONSThis article references 30 other publications.
- 1Saleh, L.; Perler, F. B. Chem. Rec. 2006, 6, 183– 93[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XptlymsLs%253D&md5=adc1e1637c25e42f418d240e1cf080e2Protein splicing In Cis and In TransSaleh, Lana; Perler, Francine B.Chemical Record (2006), 6 (4), 183-193CODEN: CRHEAK; ISSN:1527-8999. (John Wiley & Sons, Inc.)A review. Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallog. study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were detd. using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.
- 2Pietrokovski, S. Trends Genet. 2001, 17, 465– 72[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlsFylu7s%253D&md5=149124813929befcde02e4d67d8f8887Intein spread and extinction in evolutionPietrokovski, S.Trends in Genetics (2001), 17 (8), 465-472CODEN: TRGEE2; ISSN:0168-9525. (Elsevier Science Ltd.)A review. Inteins are selfish DNA elements found within coding regions. They are translated with their host protein, but then catalyze their own excision and the formation of a peptide bond between their flanking protein regions. Understanding what drives and selects inteins is relevant for assessing whether they have unidentified biol. functions and whether they can invade and become established in new genes and organisms. Inteins are suggested to have been present and more common in the progenitors of eukaryotes and prokaryotes. In these cells, inteins had some beneficial function or had evolved from an unknown beneficial protein. Since then, this putative benefit has been lost and inteins are gradually becoming extinct. The proteins in which inteins are currently found are proposed to be proteins vital for the survival of the organism, where intein removal is most difficult.
- 3Perler, F. B. Nucleic Acids Res. 2002, 30, 383– 4[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xht12rsb0%253D&md5=fa2463b3bd0fbdd25f28cba1fba0fe51InBase: the Intein DatabasePerler, Francine B.Nucleic Acids Research (2002), 30 (1), 383-384CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliog. An intein-specific BLAST server is now available to assist in identifying new inteins.
- 4Vila-Perelló, M.; Muir, T. W. Cell 2010, 143, 191– 200[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht12qurnM&md5=12f45692e4a8d7b87e72799d03a300abBiological Applications of Protein SplicingVila-Perello, Miquel; Muir, Tom W.Cell (Cambridge, MA, United States) (2010), 143 (2), 191-200CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a mol. disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chem. and biol., allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.
- 5Caspi, J.; Amitai, G.; Belenkiy, O.; Pietrokovski, S. Mol. Microbiol. 2003, 50, 1569– 77[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVShtb3I&md5=6f713ce6753313b31ff104f4fc6f4459Distribution of split DnaE inteins in cyanobacteriaCaspi, Jonathan; Amitai, Gil; Belenkiy, Olga; Pietrokovski, ShmuelMolecular Microbiology (2003), 50 (5), 1569-1577CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analyzing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnol.
- 6Dassa, B.; London, N.; Stoddard, B. L.; Schueler-Furman, O.; Pietrokovski, S. Nucleic Acids Res. 2009, 37, 2560– 73[Crossref], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlsleru7c%253D&md5=47ca9d9d02a3be15d575b019473e0e5aFractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease familyDassa, Bareket; London, Nir; Stoddard, Barry L.; Schueler-Furman, Ora; Pietrokovski, ShmuelNucleic Acids Research (2009), 37 (8), 2560-2573CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic anal. of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this fractured' organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) endonucleases, was found in half of the loci. These putative homing endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.
- 7Southworth, M. W.; Adam, E.; Panne, D.; Byer, R.; Kautz, R.; Perler, F. B. EMBO J. 1998, 17, 918– 26[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhs1yrtLk%253D&md5=be87a8099c13aff8d8ce7d8ddd3a2120Control of protein splicing by intein fragment reassemblySouthworth, Maurice W.; Adam, Eric; Panne, Daniel; Byer, Robyn; Kautz, Roger; Perler, Francine B.EMBO Journal (1998), 17 (4), 918-926CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in sep. hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endo-nuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or contg. affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not obsd. with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR anal. or enabling the study of the effects of any type of protein modification on a limited region of a protein.
- 8Yamazaki, T.; Otomo, T.; Oda, N.; Kyogoku, Y.; Uegaki, K.; Ito, N.; Ishino, Y.; Nakamura, H. J. Am. Chem. Soc. 1998, 120, 5591– 5592[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtlWku7o%253D&md5=df604b078a8bcd7ffc9d4293a151b694Segmental Isotope Labeling for Protein NMR Using Peptide SplicingYamazaki, Toshio; Otomo, Takanori; Oda, Natsuko; Kyogoku, Yoshimasa; Uegaki, Koichi; Ito, Nobutoshi; Ishino, Yoshizumi; Nakamura, HarukiJournal of the American Chemical Society (1998), 120 (22), 5591-5592CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)This article demonstrates a new method for the ligation of labeled and unlabeled peptide fragments using a peptide splicing element, intein (Protozyme). This method is expected to facilitate the application of NMR to proteins as large as 50 kDa. Segmental labeling will make it possible to assign signals unambiguously in a short time and to det. the structures of such large proteins to a higher precision.
- 9Mootz, H. D.; Blum, E. S.; Tyszkiewicz, A. B.; Muir, T. W. J. Am. Chem. Soc. 2003, 125, 10561– 9[ACS Full Text], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmtVKntLs%253D&md5=6626d6a79010d4cfa48a1f4e437c216cConditional protein splicing: A new tool to control protein structure and function in vitro and in vivoMootz, Henning D.; Blum, Elyse S.; Tyszkiewicz, Amy B.; Muir, Tom W.Journal of the American Chemical Society (2003), 125 (35), 10561-10569CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small mol. rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunopptn. in cells treated with rapamycin or a nontoxic analog thereof. No background splicing in the absence of the small-mol. inducer was obsd. over a 24-h time course. Product formation could be detected within 10 min of addn. of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small mol. ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.
- 10Oeemig, J. S.; Aranko, A. S.; Djupsjöbacka, J.; Heinämäki, K.; Iwaï, H. FEBS Lett. 2009, 583, 1451– 1456[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlsVKgsbw%253D&md5=1985910fe023b377877ccca6c61e175cSolution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site-specific chemical modificationOeemig, Jesper S.; Aranko, A. Sesilja; Djupsjoebacka, Janica; Heinaemaeki, Kimmo; Iwai, HideoFEBS Letters (2009), 583 (9), 1451-1456CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We detd. the soln. structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.
- 11Shi, J.; Muir, T. W. J. Am. Chem. Soc. 2005, 127, 6198– 206[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjtVWntr4%253D&md5=f2eb1818bd9e802e55faace23424dbecDevelopment of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split InteinsShi, Jianxin; Muir, Tom W.Journal of the American Chemical Society (2005), 127 (17), 6198-6206CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chem. biol. and biotechnol., precious little is known about the mol. recognition events governing the initial fragment assocn. step. In this study, fluorescence approaches have been used to measure the dissocn. const. for the Ssp DnaE split intein interaction and to det. the on and off rates of fragment assocn. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid assocn. of the fragments at physiol. pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technol. should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins.
- 12Shah, N. H.; Vila-Perelló, M.; Muir, T. W. Angew. Chem., Int. Ed. 2011, 50, 6511– 5[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnt1eitbg%253D&md5=98573036657447152bc6a05d5a37adb9Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split inteinShah, Neel H.; Vila-Perello, Miquel; Muir, Tom W.Angewandte Chemie, International Edition (2011), 50 (29), 6511-6515, S6511/1-S6511/45CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)While ionic interactions have previously been postulated to play a role in split intein assembly, the involvement of electrostatic forces has not been validated exptl. The authors set out to test the hypothesis that ionic interactions facilitate the assocn. of split intein fragments and thus could be manipulated to control the relative reactivities of different N- and C-intein complexes. This study probed the role of intermol. ion clusters for fragment assembly and splicing in the NpuWT split intein both in vivo and in vitro. Through these expts., the authors rationally designed a new split intein, NpuMUT, which displays low cross-reactivity with NpuWT. These orthogonal inteins were used to generate the large, full-length, active mammalian protein PARP1 through a one-pot three-piece ligation. Collectively, the results demonstrate that electrostatic interactions can engender kinetic control in a complex enzymic system. Furthermore, these results provide insight into the mol. requirements for efficient and specific protein trans-splicing.
- 13Zheng, Y.; Wu, Q.; Wang, C.; Xu, M.-Q.; Liu, Y. Biosci. Rep. 2012, 32, 433– 42[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlCjsb3N&md5=86818bead0373351bc14a6416f4fe33cMutual synergistic protein folding in split inteinZheng, Yuchuan; Wu, Qin; Wang, Chunyu; Xu, Min-qun; Liu, YangzhongBioscience Reports (2012), 32 (5), 433-442CODEN: BRPTDT; ISSN:0144-8463. (Portland Press Ltd.)Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two sepd. halves, and the recognition and assocn. of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodn. anal. on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon assocn. ITC (isothermal titrn. calorimetry) reveals that the highly favorable enthalpy change drives the assocn. of the two halves, overcoming the unfavorable entropy change. The high flexibility of two fragments and the marked thermodn. preference provide a robust assocn. for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein.
- 14Zettler, J.; Schütz, V.; Mootz, H. D. FEBS Lett. 2009, 583, 909– 14[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXis1aqtb0%253D&md5=6ee44ff008bd1d4d44f73c960671b716The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reactionZettler, Joachim; Schuetz, Vivien; Mootz, Henning D.FEBS Letters (2009), 583 (5), 909-914CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)We have studied the naturally split α subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and detd. an apparent first-order rate const. of (1.1 ± 0.2) × 10-2 s-1 at 37 °C. This represents the highest rate reported to date for the protein trans-splicing reaction (t1/2 of ∼60 s). Furthermore, the reaction was very robust and exhibited high yield with respect to extein sequence, temp. (6 to 37°C), and urea (up to 6 M). Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chem.
- 15Shah, N. H.; Dann, G. P.; Vila-Perelló, M.; Liu, Z.; Muir, T. W. J. Am. Chem. Soc. 2012, 134, 11338– 41[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptF2iu7Y%253D&md5=dd6b4d96222fde82854c206d01128851Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein EngineeringShah, Neel H.; Dann, Geoffrey P.; Vila-Perello, Miquel; Liu, Zhihua; Muir, Tom W.Journal of the American Chemical Society (2012), 134 (28), 11338-11341CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly obsd. for this autoprocessing protein family. Furthermore, when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chem. biol.
- 16Muona, M.; Aranko, A. S.; Raulinaitis, V.; Iwaï, H. Nat. Protoc. 2010, 5, 574– 87[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislOmsLs%253D&md5=b5dbc0b472c9ae567a8ebcd79bcb5ac1Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitroMuona, Mikko; Aranko, A. Sesilja; Raulinaitis, Vytas; Iwai, HideoNature Protocols (2010), 5 (3), 574-587CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Segmental isotopic labeling is a powerful labeling technique for reducing NMR signal overlap, which is assocd. with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR expts. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or α-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total prepn. time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.
- 17Borra, R.; Dong, D.; Elnagar, A. Y.; Woldemariam, G. A.; Camarero, J. A. J. Am. Chem. Soc. 2012, 134, 6344– 53[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1WksrY%253D&md5=578e1f4afc767f2f0844dd66320e5017In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split InteinsBorra, Radhika; Dong, Dezheng; Elnagar, Ahmed Y.; Woldemariam, Getachew A.; Camarero, Julio A.Journal of the American Chemical Society (2012), 134 (14), 6344-6353CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Methods to visualize, track, and modify proteins in living cells are central for understanding the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chem. labeling of proteins. The authors report in this work the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. The authors have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, the authors have shown that this approach can be also used for modifying proteins to control their cellular localization and potentially alter their biol. activity.
- 18Vila-Perelló, M.; Liu, Z.; Shah, N. H.; Willis, J. A.; Idoyaga, J.; Muir, T. W. J. Am. Chem. Soc. 2013, 135, 286– 92[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvV2gsL3E&md5=ea910b6e58fc18fa6f5da446d117af1aStreamlined Expressed Protein Ligation Using Split InteinsVila-Perello, Miquel; Liu, Zhihua; Shah, Neel H.; Willis, John A.; Idoyaga, Juliana; Muir, Tom W.Journal of the American Chemical Society (2013), 135 (1), 286-292CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. modified proteins are invaluable tools for studying the mol. details of biol. processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-contg. peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. The authors describe here a new method for the prepn. and purifn. of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to assoc. to become active. Taking advantage of the strong affinity between the two split intein fragments, the authors devised a streamlined procedure for the purifn. and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.
- 19Jagadish, K.; Borra, R.; Lacey, V.; Majumder, S.; Shekhtman, A.; Wang, L.; Camarero, J. A. Angew. Chem., Int. Ed. 2013, 52, 3126– 31[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXos1agug%253D%253D&md5=f32c6c42f1a3c7783a8c737117dd07f4Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide-Protein InteractionsJagadish, Krishnappa; Borra, Radikha; Lacey, Vanessa; Majumder, Subhabrata; Shekhtman, Alexander; Wang, Lei; Camarero, Julio A.Angewandte Chemie, International Edition (2013), 52 (11), 3126-3131CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors showed that the biosynthesis of cyclotides contg. non-natural amino acids can be achieved by using different intein-based methods. EPL-backbone cyclization can provide non-natural amino acid contg. cyclotides when the cyclization is carried out in vitro by GSH-induced cyclization and folding of the corresponding precursor. In vivo prodn., however, is less efficient using this method. It was shown that PTS-mediated backbone cyclization using the highly efficient Npu DnaE split-intein can be used for the efficient prodn. of cyclotides inside live E. coli cells. It was estd. that the in vivo prodn. of MCoTI-I was around seven times more efficient using Npu DnaE PTS than EPL, thereby providing an attractive alternative for the prodn. of these types of polypeptides in bacterial cells. The high efficiency of PTS-mediated cyclization combined with nonsense-suppressing orthogonal tRNA/synthetase technol. made the in vivo prodn. of cyclotides contg. non-natural amino acids possible. Of particular interest is the introduction of azido-contg. amino acids, which can react with DBCO-contg. fluorescent probes to provide in vivo fluorescently labeled cyclotides. Cyclotides contg. the nonnatural amino acid AziF can be expressed in live bacterial cells and easily labeled with DBCO-AMCA to monitor cyclotide-protein interactions. This finding opens the possibility for in vitro and potentially also in vivo screening of genetically encoded libraries of cyclotides for the rapid selection of novel cyclotide sequences able to bind a specific bait protein using high-throughput cell-based optical screening approaches.
- 20Sorci, M.; Dassa, B.; Liu, H.; Anand, G.; Dutta, A. K.; Pietrokovski, S.; Belfort, M.; Belfort, G. Anal. Chem. 2013, 85, 6080– 8[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvVCqtrk%253D&md5=5a745992f179e62da10f2310600d7c0eOriented Covalent Immobilization of Antibodies for Measurement of Intermolecular Binding Forces between Zipper-Like Contact Surfaces of Split InteinsSorci, Mirco; Dassa, Bareket; Liu, Hongwei; Anand, Gaurav; Dutta, Amit K.; Pietrokovski, Shmuel; Belfort, Marlene; Belfort, GeorgesAnalytical Chemistry (Washington, DC, United States) (2013), 85 (12), 6080-6088CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In order to measure the intermol. binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermol. force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (∼52 pN). Bioinformatic sequence anal. and computational structural anal. discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, the authors tested the Jarzynski's equality and demonstrated, as expected, that nonequil. dissipative measurements obtained here gave larger energies of interaction as compared with those for equil. Hence, AFM coupled with the authors' immobilization strategy and computational studies provides a useful anal. tool for the direct measurement of intermol. assocn. of split inteins and could be extended to any interacting protein pair.
- 21Shah, N. H.; Muir, T. W. Isr. J. Chem. 2011, 51, 854– 861[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2gsbzP&md5=c51573533e69199e56a4de578d92981fSplit inteins: Nature's protein ligasesShah, Neel H.; Muir, Tom W.Israel Journal of Chemistry (2011), 51 (8-9), 854-861CODEN: ISJCAT; ISSN:0021-2148. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Split inteins carry out a naturally occurring process known as protein trans-splicing, where 2 protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past 13 yr since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a no. of biotechnol. applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, the authors discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.
- 22Shah, N. H.; Eryilmaz, E.; Cowburn, D.; Muir, T. W. J. Am. Chem. Soc. 2013, 135, 5839– 47[ACS Full Text], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktFKgtLg%253D&md5=0b249056e530bcd593e886755ab66332Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-SplicingShah, Neel H.; Eryilmaz, Ertan; Cowburn, David; Muir, Tom W.Journal of the American Chemical Society (2013), 135 (15), 5839-5847CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Split inteins play an important role in modern protein semi-synthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic redn. in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here a thorough kinetic anal. of the relationship between C-extein compn. and split intein activity is presented. The results of these expts. were used to guide structural and mol. dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, the structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.
- 23Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Proteins: Struct., Funct., Genet. 2000, 41, 415– 427[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnslGnsLs%253D&md5=ddd4a8ba1d035c3d566e1a50b509a173Why are "Natively unfolded" proteins unstructured under physiologic conditions?Uversky, Vladimir N.; Gillespie, Joel R.; Fink, Anthony L.Proteins: Structure, Function, and Genetics (2000), 41 (3), 415-427CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)"Natively unfolded" proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Anal. of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and "natively unfolded" ones. The results show that "natively unfolded" proteins are specifically localized within a unique region of charge-hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of "natively unfolded" proteins.
- 24Müller-Späth, S.; Soranno, A.; Hirschfeld, V.; Hofmann, H.; Rüegger, S.; Reymond, L.; Nettels, D.; Schuler, B. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14609– 14[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVOgsb7K&md5=8b1083dd092c95fba89c18bf4b70394dCharge interactions can dominate the dimensions of intrinsically disordered proteinsMuller-Spath, Sonja; Soranno, Andrea; Hirschfeld, Verena; Hofmann, Hagen; Ruegger, Stefan; Reymond, Luc; Nettels, Daniel; Schuler, BenjaminProceedings of the National Academy of Sciences of the United States of America (2010), 107 (33), 14609-14614, S14609/1-S14609/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many eukaryotic proteins are disordered under physiol. conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-mol. Forster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been obsd. for many proteins at low denaturant concn., IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an addnl. collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.
- 25Semisotnov, G. V.; Rodionova, N. A.; Razgulyaev, O. I.; Uversky, V. N.; Gripas, A. F.; Gilmanshin, R. I. Biopolymers 1991, 31, 119– 128[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhvVWmurY%253D&md5=e0d4a2d45d8d8482cfdc1d21b15eebfeStudy of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probeSemisotnov, G. V.; Rodionova, N. A.; Razgulyaev, O. I.; Uverskii, V. N.; Gripas, A. F.; Gil'manshin, R. I.Biopolymers (1991), 31 (1), 119-28CODEN: BIPMAA; ISSN:0006-3525.Binding of the hydrophobic fluorescent probe 1-anilino-naphthalene-8-sulfonate (ANS) to synthetic polypeptides and proteins with a different structural organization was studied. It was shown that ANS has a much stronger affinity to the protein molten globule state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, α-helical, or β-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equil. and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, β-lactamase, and α-lactalbumin as examples.
- 26Xu, R.; Ayers, B.; Cowburn, D.; Muir, T. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 388– 93[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmtlCgsg%253D%253D&md5=b44cd6f792c3957b0c5b7dfc59807b2fChemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studiesXu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (2), 388-393CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A convenient in vitro chem. ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homol. type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory app. of the protein, were individually prepd. in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prep. NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prep. recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chem. ligation in vitro, will increase the size limits for protein structural detn. in soln. with NMR methods. In vitro chem. ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
- 27Millet, O.; Loria, J. P.; Kroenke, C. D.; Pons, M.; Palmer, A. G. J. Am. Chem. Soc. 2000, 122, 2867– 2887[ACS Full Text], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Crsbs%253D&md5=d2e62cf97ded2433edaebae5b7ffe704The Static Magnetic Field Dependence of Chemical Exchange Linebroadening Defines the NMR Chemical Shift Time ScaleMillet, Oscar; Loria, J. Patrick; Kroenke, Christopher D.; Pons, Miquel; Palmer, Arthur G., IIIJournal of the American Chemical Society (2000), 122 (12), 2867-2877CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The static magnetic field dependence of chem. exchange linebroadening in NMR spectroscopy is investigated theor. and exptl. Two-site exchange (A ↹ B) is considered with site A more highly populated than site B (pa > pb), a shift difference between sites equal to Δω, and an exchange rate const. given by kex. The exchange contribution to the transverse relaxation rate const. for the more highly populated site is denoted Rex. The dependence of Rex on the static magnetic field strength is characterized by a scaling parameter α = d ln Rex/d ln Δω, in which 0 ≤ α ≤ 2 for pa > 0.7. The value of α depends on the NMR chem. shift time scale for the exchange process: for slow exchange (kex/Δω < 1), 0 ≤ α < 1; for intermediate exchange (kex/Δω = 1), α = 1; and for fast exchange (kex/Δω > 1), 1 < α ≤ 2. Consequently, the static magnetic field dependence of Rex defines the chem. shift time scale for an exchange process even if the populations are so highly skewed (pa » pb) that the minor resonance is not observable in the slow exchange limit. The theor. results are verified by measuring 15N transverse relaxation rate consts. at static magnetic fields of 11.7 and 14.1 T and temps. of 300 and 313 K for the protein basic pancreatic trypsin inhibitor. At each combination of static magnetic field and temp., the rate consts. were measured using Carr-Purcell-Meiboom-Gill and Hahn echo techniques with spin-echo delays ranging from 1.0 to 64.5 ms. 15N resonances for residues in the region of the Cys14-Cys38 disulfide bond are broadened due to chem. exchange. Values of α obtained from the relaxation rate consts. range from 0.26 ± 0.17 for Arg39 at 300 K to 1.96 ± 0.25 for Cys38 at 313 K. For Cys38 and Arg39, the two residues most strongly affected by chem. exchange, values of kex were detd. to be 380 ± 70 s-1 and 530 ± 90 s-1 at 300 K and 1300 ± 290 s-1 and 1370 ± 160 s-1 at 313 K by global anal. of the relaxation rate consts. The scaling parameters α indicate that chem. exchange for most residues in basic pancreatic trypsin inhibitor does not satisfy kex/Δω » 1. Consequently, the assumption of fast-limit quadratic scaling of exchange broadening in proteins and other macromols. may be incorrect, even if a single broadened resonance is obsd. for a nuclear spin. The theor. results for the static magnetic field dependence of chem. exchange broadening in NMR spectroscopy are applicable to other nuclei and to other techniques for measuring chem. exchange linebroadening.
- 28Shoemaker, B. A.; Portman, J. J.; Wolynes, P. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8868– 73[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXls12ltLY%253D&md5=614acf652b32372b4d90f53fb64070a8Speeding molecular recognition by using the folding funnel: the fly-casting mechanismShoemaker, Benjamin A.; Portman, John J.; Wolynes, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (16), 8868-8873CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein mol. can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the "fly-casting mechanism.". We illustrate this scenario with the hypothetical kinetics of binding a single repressor mol. to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.
- 29Schreiber, G.; Fersht, A. R. Nat. Struct. Biol. 1996, 3, 427– 31[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XivVahtrk%253D&md5=f2b4e71b355b469c425d055f4d9562f3Rapid, electrostatically assisted association of proteinsSchreiber, Gideon; Fersht, Alan R.Nature Structural Biology (1996), 3 (5), 427-431CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)The rapid assocn. of barnase and its intracellular inhibitor barstar has been analyzed from the effects of mutagenesis and electrostatic screening. A basal assocn. rate const. of 105 M-1 s-1 is increased to over 5 × 109 M-1 s-1 by electrostatic forces. The assocn. between the oppositely charged proteins proceeds through the rate-detg. formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase assocn. rate consts. between mols. and its principles may be used for protein design.
- 30Choi, J. J.; Nam, K. H.; Min, B.; Kim, S.-J.; Söll, D.; Kwon, S.-T. J. Mol. Biol. 2006, 356, 1093– 106[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlGmtr8%253D&md5=c62f01ce7e79634f7c219705ab343487Protein Trans-splicing and Characterization of a Split Family B-type DNA Polymerase from the Hyperthermophilic Archaeal Parasite Nanoarchaeum equitansChoi, Jeong Jin; Nam, Ki Hoon; Min, Bokkee; Kim, Sang-Jin; Soell, Dieter; Kwon, Suk-TaeJournal of Molecular Biology (2006), 356 (5), 1093-1106CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two sep. genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80° after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examd. in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temp., and occurred only at temps. above 50°. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'→5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'→5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show exptl. that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.
Supporting Information
ARTICLE SECTIONSFull methods and experimental data, including protein semisynthesis and purification protocols, characterization of proteins, and additional biophysical data. This material is available free of charge via the Internet at http://pubs.acs.org.
Terms & Conditions
Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html.