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β-Sheet folding mechanisms from perturbation energetics
b-Sheet folding mechanisms from perturbation energetics Songpon Deechongkit1, Houbi Nguyen2, Marcus Jager1, Evan T Powers1, Martin Gruebele2 and Jeffery W Kelly1 Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of b-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in b-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional b-sheets, consistent with evolutionary robustness. Addresses 1 Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 2 Center for Biophysics and Computational Biology, Departments of Chemistry and Physics, University of Illinois, Urbana, IL 61801, USA Corresponding author: Kelly, Jeffery W ([email protected])
Current Opinion in Structural Biology 2006, 16:94–101 This review comes from a themed issue on Folding and binding Edited by Mikael Oliveberg and Eugene I Shakhnovich Available online 25th January 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.01.014
Introduction Considerable progress has been made in understanding b-sheet folding, especially in the past decade. Increasingly sophisticated methods have been applied to extract intrinsic b-sheet propensities from protein crystal structures, beginning with the classic work of Chou and Fasman [1–3]. The protein database (PDB) has been mined for information on turn and loop structures [4,5], crossstrand sidechain–sidechain interactions [6–8] and other determinants of b-sheet stability [9]; these factors have also been studied in several kinds of model systems. Intrinsic b-sheet propensities have been determined from mutational analyses of folded peptides and proteins [10– 14]. Reverse turn propensity has been studied in many contexts and model systems [15–19,20,21,22,23]. The Current Opinion in Structural Biology 2006, 16:94–101
strength of cross-strand sidechain–sidechain interactions and the importance of hydrophobic core formation have been studied in autonomously folding b-hairpins [16,19,24–30] and small protein domains [31–33]. Even statistical mechanical models of b-sheet formation, similar to the Zimm–Bragg and Lifson–Roig models of a-helix formation, have been reported [34,35]. The information gained has also enabled the de novo design, preparation and characterization of b-hairpins and cooperatively folding b-sheets [36–44]. The classic models of protein folding (framework, hydrophobic collapse and related models) have been superseded by the energy landscape or folding funnel hypothesis (Figure 1) [45,46]. The energy landscape hypothesis proposes that protein folding can be visualized as occurring on a multidimensional surface, usually depicted in three dimensions. On this surface, the ensemble of conformations that can be regarded as native is represented by a point of lowest energy. Points on the surface further and further away from the native state represent conformations that are less and less native like. When the conformations on the surface are plotted against energy, the surface takes on a funnel-like shape, with the native state at the bottom of the funnel (as it is presumably the lowest energy conformation). The folding funnel concept predicts that a b-sheet fold can be accessed through numerous pathways and that transitions between individual conformations during b-sheet folding could occur with modest barriers (kT), given an ideal sequence. In such cases, the folding funnel is said to be smooth. Our data on WW domains (Figure 2) are consistent with both of these predictions, vide infra. WW domains comprise triple-stranded b-sheets with two intervening loops. They bind proline-rich sequences, which are sometimes phosphorylated, and mediate protein–protein interactions involved in signal transduction and other biological functions [47,48]. This large family of 40 amino acid domains can be produced by either solidphase chemical synthesis or recombinant expression, enabling diverse structural modifications to be made [22,23,33,49–53,54]. Importantly, WW domains are amenable to extensive mutagenesis, typically with retention of the native fold [55]. In this review, we will discuss how perturbations of both the WW domain backbone and sidechains, in conjunction with information from the evolutionary record, have been used to understand WW domain folding. www.sciencedirect.com
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Figure 1
Folding funnels for the PIN WW domain consistent with experimental data. The x- and y-axes represent hypothetical conformational coordinates. The z-axis represents energy. (a) When the folding funnel is relatively smooth, the protein can use many paths to fold and there is no clearly defined TS. (b) When the folding funnel is rougher, the folding routes are biased so that different starting points (i.e. different unfolded structures) pass through the same or similar rate-limiting TS. For the PIN WW domain, loop 1 is structured in the TS.
Thermodynamic determinants of a three-stranded b-sheet Traditional mutagenesis of proteins results in sidechain alteration while maintaining backbone structure in most cases, whereas amide to ester (A-to-E) and amide to Eolefin (A-to-O) mutations perturb backbone structure while preserving sidechain structure (Figure 3) [56–62]. Backbone mutagenesis typically eliminates one (A-to-E) or two (A-to-O) hydrogen bonds. Traditional mutagenesis reveals that the majority of the sidechains of the PIN WW domain can be mutated without substantial destabilization of the fold [22,23,33,51–53]. The exceptions are the blue sidechains shown in Figure 2, which, when mutated, substantially destabilize the WW domain because they form the small but important hydrophobic core of this family of proteins. Perhaps surprisingly, an A-to-E backbone perturbation scan of the PIN WW domain reveals that eliminating the majority of hydrogen-bond donors one at a time does not substantially destabilize the WW domain [54,63]. A-to-E mutations lead to unfolded WW domains only when they eliminate hydrogen bonds located within the hydrophobic core. This implies that hydrogen bonds enveloped by a hydrophobic core contribute more to stability (colored in red in Figure 2) than those with partial or full exposure to the aqueous environment [54,63]. Interestingly, there are three residues in the PIN WW domain (Tyr23, Phe25, www.sciencedirect.com
Asn26) that contribute both energetically important hydrogen bonds and sidechains to the hydrophobic core, and disproportionately contribute to the stability of the domain. However, this stability core is not to be confused with a folding nucleus, which would influence the rate of folding [22,33,51,54,63]. Of course, hydrogen bonds are electrostatic interactions and it is reasonable that those in a low dielectric region of the protein (in the hydrophobic core) would contribute more to stability than those in a solvent-exposed high dielectric region. It remains unclear what subset of molecular information is necessary and sufficient to achieve a b-sheet fold. However, from the perspective of stability alone (not yet considering the pathways by which the fold is acquired, vide infra), it is clear that only a subset of the dense network of interatomic interactions observed in protein structures is required [22,23,33,51–53,54,63,64,65]. This suggests that proteins could maintain their native state even after extensive sidechain and backbone mutagenesis, as long as the critical subset of interactions was maintained. This observation, if it proves to be general, which we expect to be the case, could enable proteins to be substantially modified without compromising structure or function for applications including pharmaceutical uses. Current Opinion in Structural Biology 2006, 16:94–101
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Figure 2
Structure of the PIN WW domain. (a) Cartoon of the PIN WW domain structure, indicating residues important for the TS structure and energetics (and therefore the folding rate) in red, and residues important for native state thermodynamic stability in blue. Arrows indicate hydrogen bonds, with the arrowheads pointing towards the acceptor. (b) Structure of the PIN WW domain backbone, with the residues, strands and loops labeled. Residues with sidechains in the hydrophobic core are identified as blue ovals. Buried hydrogen bonds are shown in red, whereas solvent-exposed hydrogen bonds are shown in blue. (c) PIN WW domain structure, with the surface of the hydrophobic core shown in transparent blue and the energetically important hydrogen bonds shown in red. Adapted from [54].
As stated above, the folding funnel hypothesis implies that the acquisition of the native state could occur by many pathways in cases in which the folding funnel is relatively smooth (Figure 1a), making it very difficult to
understand the structural requirements for the nucleation of b-sheet folding [46]. By contrast, roughness in the energy landscape (i.e. energetic barriers substantially larger than kT between individual conformations) would
Figure 3
The effect of A-to-E and A-to-O mutations on hydrogen bonding. An A-to-E mutation weakens one hydrogen bond (as the ester carbonyl is a weaker hydrogen-bond acceptor than an amide carbonyl) and eliminates one hydrogen bond (because the non-carbonyl ester oxygen cannot donate a hydrogen bond). The eliminated hydrogen bond is replaced by modest electrostatic repulsion between the non-carbonyl ester oxygen and the adjacent amide carbonyl. An A-to-O mutation simply removes two hydrogen bonds. Current Opinion in Structural Biology 2006, 16:94–101
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bias the routes taken by most proteins down the folding funnel, such that a family of related pathways or mechanisms would lead to a given b-sheet fold (Figure 1b) [45]. The experimental data collected suggest that b-sheet landscapes are generally rough enough to extract structural information about the transition state (TS) from energetic analyses of mutants [66]. Although it is important to begin to think about the collection and interpretation of protein folding kinetic and thermodynamic data from the perspective of multiple folding pathways for a given sequence, this has yet to be widely embraced by experimentalists.
Kinetic mechanism of WW domain folding The differential influence of a sidechain or backbone mutation on the TS and ground state structures can be discerned using the linear free energy analysis popularized by Fersht [22,23,33,51–53,54,66]. The so-called Fvalue analysis allows one to interrogate the TS structure: FM ¼
y y DDGyf DGf;mut DGf;wt ¼ DDGf DGf;mut DGf;wt
where DGf,mut and DGf,wt are folding free energies, and DDGyf;mut and DDGyf;wt are the activation free energies (i.e.
the free energy difference between the TS and the unfolded state) of the mutant and wild-type proteins, respectively. Values of DDGyf can be extracted from measurements of the folding kinetics. The relaxation time constants exhibited by WW domains are in the 5– 200 ms range (rate constants of 104–105 s1), requiring the use of the laser T-jump method to interrogate their rapid folding [22,23,33,51–53,54]. A FM value of 1 indicates that the interactions perturbed by a mutation stabilize or destabilize the folding TS and the native state to the same extent, that is, the protein has native-like structure in the vicinity of the mutation in the TS [66]. A FM value of 0 indicates the opposite scenario; the protein is unstructured in the vicinity of the mutation in the folding TS. Intermediate values of FM are more difficult to interpret [67], but for proteins exhibiting minimal frustration in their folding landscapes, intermediate FM values represent the average extent to which perturbed interactions exist in the folding TS. Extensive traditional mutagenesis of WW domains reveals FM values of 0.2–0.6 for all secondary structure regions except loop 1, which exhibits high values (FM > 0.8), indicating the formation of loop 1 in the TS [22,33,52,53] (Figure 4a). FM values exceeding 1 are
Figure 4
Summary of PIN WW domain FM values for (a) traditional sidechain variants and (b) A-to-E mutations. Residues for which FM values have been measured are shown in bold. www.sciencedirect.com
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generally interpreted as being due to non-native interactions in the TS [68] that become non-existent at higher temperature. The FM values resulting from A-to-E backbone mutations of the WW domain are particularly informative because, unlike traditional sidechain mutations, they directly describe the hydrogen-bond-mediated formation of secondary structure in the TS in the region proximal to the mutation [54,63]. Data from an A-to-E scan of the WW domain backbone reveal that loop 1 is structured, but not yet native like in the folding TS (FM values approaching 1), consistent with the sidechain perturbation data [54] (Figure 4b). Significant progress by several laboratories studying the mechanisms of bsheet folding reveals that turn formation is often the ratelimiting step in b-sheet and b-hairpin formation, although under certain conditions hydrophobic collapse can become rate limiting [22,23,33,54,69–71,72,73,74, 75,76]. For example, the TS of WW domains can shift appreciably as the folding temperature is varied from 37 8C [33,54,75]. The high FM values exhibited by loop 1 of PIN WW domains (Figure 4) imply that WW domain folding could become faster if the six-residue loop with an internal bturn was transformed into a five- or four-residue turn with a sequence optimized to reverse the direction of the polypeptide chain while maintaining the right-handed twist found in the majority of WW domains. Grafting optimal turn sequences into a PIN host sequence in lieu of loop 1 affords the fastest folding WW domains characterized to date, exhibiting folding time constants of 5 ms, within an order of magnitude of the time constants exhibited by b-hairpins [23]. Interestingly, grafting these fast folding shorter sequences reveals that loop 1 now also substantially influences thermodynamic stability. This effort demonstrates that loop engineering can lower the TS energy by more than 1 kcal/mol (as determined from a two-state Kramers analysis), transforming the WW domain into a near-downhill folder, that is, a sequence that folds into a b-sheet with barriers <0.5 kcal/mol and therefore exhibits a relatively smooth folding funnel (Figure 1a). The evolutionary record of the 120 WW domain family members can be mined to understand the requirements for structure and function [64,65]. Ranganathan and colleagues interrogated each position of the WW domain family with regard to the deviation of the amino acid distribution at that position from the mean distribution found in all proteins, allowing the identification of conserved residues [64,65]. They next identified residue positions exhibiting co-evolution. It is interesting to compare those residues deemed to be important from a co-evolution perspective with those analogously classified as being important on energetic grounds. With regard to fold stability, only two residues, Phe25 and Asn26 (numbering as in this review), have been flagged as important Current Opinion in Structural Biology 2006, 16:94–101
by both perturbation energetic measurements (mutagenesis) and Ranganathan’s statistical coupling analysis [64,65]. Totally or very highly conserved positions, such as Trp11, Tyr23, Trp34 and Pro37, must be formally treated as having the potential for co-evolution, but the lack of variation at these positions precludes the analysis used by Ranganathan. In the case of the two invariant residues for which these domains are named, previous mutagenesis data demonstrate that Trp11 appears to be crucial for thermodynamic stability, whereas Trp34 appears to be important for function [49]. Mutagenesis of residues Glu12, His27 and Ser32, identified as being important from a co-evolution perspective, only modestly alters energetics, indicating that these residues are probably important for WW domain function or dynamics, but do not principally influence folding energetics. The statistical coupling analysis matrix for assessing the co-evolution of residues is very powerful in that it allows hypotheses about the evolutionary constraints on a protein to be tested experimentally. The energetics of folding and function may manifest as statistical couplings, and it will be interesting to discern from future studies whether these and other features are distinct, overlapping or inseparable features of the total sequence information. The coevolution data, combined with Ranganathan’s ability to computationally design WW domains that fold and function by retaining conserved and co-evolving residues only (no tertiary structure information is used), indicate that a relatively small subset of the molecular interactions observed in WW domain structures may be sufficient for folding and function [64,65]. It remains to be discerned whether this is the case for other b-sheet folds and proteins in general, although we expect that, in the case of larger proteins, this small set of interactions could be distributed throughout the sequence. The advantage of studying the evolutionary record without being biased by structural information is that it has the potential to reveal critical residues in b-sheets that perform functions in proteins that we do not currently understand.
Conclusions The combined use of A-to-E and sidechain mutagenesis reinforces the take-home message that loop or reverse turn formation is rate limiting in b-sheet folding. The data obtained from these perturbations also highlights that only a subset of the molecular interactions observed in a protein structure, namely those comprising the hydrophobic core and facilitating buried hydrogen-bond formation, contributes significantly to the thermodynamics of b-sheet formation. This information, along with information mined from the evolutionary record, offers the potential to design proteins with primary structures that only modestly resemble those of naturally occurring proteins, but are nevertheless folded and functional. www.sciencedirect.com
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temperature-jump spectroscopy. Proc Natl Acad Sci USA 2004, 101:4077-4082. 72. Du D, Zhu YJ, Huang CY, Gai F: Understanding the key factors that control the rate of beta-hairpin folding. Proc Natl Acad Sci USA 2004, 101:15915-15920. The authors used elegant IR T-jump kinetics methods to provide further evidence that turn formation is rate limiting in hairpin folding. They show that a conformationally optimized turn increases the folding rate, whereas a stronger hydrophobic cluster increases hairpin stability by decreasing the unfolding rate. 73. Riddle DS, Grantcharova VP, Santiago JV, Alm E, Ruczinski I, Baker D: Experiment and theory highlight role of native state topology in SH3 folding. Nat Struct Biol 1999, 6:1016-1024. 74. Dyer RB, Maness SJ, Peterson ES, Franzen S, Fesinmeyer RM, Andersen NH: The mechanism of beta-hairpin formation. Biochemistry 2004, 43:11560-11566.
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This paper demonstrates that shorter turns, which reduce the entropic penalty for loop formation, afford the fastest folding hairpins. For threestranded sheets, this is also generally true; however, five-residue turn sequences often fold faster than b-turns (see [23]). 75. Dyer RB, Maness SJ, Franzen S, Fesinmeyer RM, Olsen KA, Andersen NH: Hairpin folding dynamics: the cold-denatured state is predisposed for rapid refolding. Biochemistry 2005, 44:10406-10415. 76. Chen RPY, Huang JJT, Chen HL, Jan H, Velusamy M, Lee CT, Fann WS, Larsen RW, Chan SI: Measuring the refolding of beta-sheets with different turn sequences on a nanosecond time scale. Proc Natl Acad Sci USA 2004, 101:7305-7310. A photolabile cage strategy was used in combination with laser-flash photolysis and photoacoustic calorimetry to demonstrate that turn formation is rate limiting in hairpin formation. Even very subtle changes in sequence can substantially alter folding rates.
Current Opinion in Structural Biology 2006, 16:94–101
Current Opinion in Structural Biology 2006, 16:94–101 This review comes from a themed issue on Folding and binding Edited by Mikael Oliveberg and Eugene I Shakhnovich Available online 25th January 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.01.014
Introduction Considerable progress has been made in understanding b-sheet folding, especially in the past decade. Increasingly sophisticated methods have been applied to extract intrinsic b-sheet propensities from protein crystal structures, beginning with the classic work of Chou and Fasman [1–3]. The protein database (PDB) has been mined for information on turn and loop structures [4,5], crossstrand sidechain–sidechain interactions [6–8] and other determinants of b-sheet stability [9]; these factors have also been studied in several kinds of model systems. Intrinsic b-sheet propensities have been determined from mutational analyses of folded peptides and proteins [10– 14]. Reverse turn propensity has been studied in many contexts and model systems [15–19,20,21,22,23]. The Current Opinion in Structural Biology 2006, 16:94–101
strength of cross-strand sidechain–sidechain interactions and the importance of hydrophobic core formation have been studied in autonomously folding b-hairpins [16,19,24–30] and small protein domains [31–33]. Even statistical mechanical models of b-sheet formation, similar to the Zimm–Bragg and Lifson–Roig models of a-helix formation, have been reported [34,35]. The information gained has also enabled the de novo design, preparation and characterization of b-hairpins and cooperatively folding b-sheets [36–44]. The classic models of protein folding (framework, hydrophobic collapse and related models) have been superseded by the energy landscape or folding funnel hypothesis (Figure 1) [45,46]. The energy landscape hypothesis proposes that protein folding can be visualized as occurring on a multidimensional surface, usually depicted in three dimensions. On this surface, the ensemble of conformations that can be regarded as native is represented by a point of lowest energy. Points on the surface further and further away from the native state represent conformations that are less and less native like. When the conformations on the surface are plotted against energy, the surface takes on a funnel-like shape, with the native state at the bottom of the funnel (as it is presumably the lowest energy conformation). The folding funnel concept predicts that a b-sheet fold can be accessed through numerous pathways and that transitions between individual conformations during b-sheet folding could occur with modest barriers (kT), given an ideal sequence. In such cases, the folding funnel is said to be smooth. Our data on WW domains (Figure 2) are consistent with both of these predictions, vide infra. WW domains comprise triple-stranded b-sheets with two intervening loops. They bind proline-rich sequences, which are sometimes phosphorylated, and mediate protein–protein interactions involved in signal transduction and other biological functions [47,48]. This large family of 40 amino acid domains can be produced by either solidphase chemical synthesis or recombinant expression, enabling diverse structural modifications to be made [22,23,33,49–53,54]. Importantly, WW domains are amenable to extensive mutagenesis, typically with retention of the native fold [55]. In this review, we will discuss how perturbations of both the WW domain backbone and sidechains, in conjunction with information from the evolutionary record, have been used to understand WW domain folding. www.sciencedirect.com
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Figure 1
Folding funnels for the PIN WW domain consistent with experimental data. The x- and y-axes represent hypothetical conformational coordinates. The z-axis represents energy. (a) When the folding funnel is relatively smooth, the protein can use many paths to fold and there is no clearly defined TS. (b) When the folding funnel is rougher, the folding routes are biased so that different starting points (i.e. different unfolded structures) pass through the same or similar rate-limiting TS. For the PIN WW domain, loop 1 is structured in the TS.
Thermodynamic determinants of a three-stranded b-sheet Traditional mutagenesis of proteins results in sidechain alteration while maintaining backbone structure in most cases, whereas amide to ester (A-to-E) and amide to Eolefin (A-to-O) mutations perturb backbone structure while preserving sidechain structure (Figure 3) [56–62]. Backbone mutagenesis typically eliminates one (A-to-E) or two (A-to-O) hydrogen bonds. Traditional mutagenesis reveals that the majority of the sidechains of the PIN WW domain can be mutated without substantial destabilization of the fold [22,23,33,51–53]. The exceptions are the blue sidechains shown in Figure 2, which, when mutated, substantially destabilize the WW domain because they form the small but important hydrophobic core of this family of proteins. Perhaps surprisingly, an A-to-E backbone perturbation scan of the PIN WW domain reveals that eliminating the majority of hydrogen-bond donors one at a time does not substantially destabilize the WW domain [54,63]. A-to-E mutations lead to unfolded WW domains only when they eliminate hydrogen bonds located within the hydrophobic core. This implies that hydrogen bonds enveloped by a hydrophobic core contribute more to stability (colored in red in Figure 2) than those with partial or full exposure to the aqueous environment [54,63]. Interestingly, there are three residues in the PIN WW domain (Tyr23, Phe25, www.sciencedirect.com
Asn26) that contribute both energetically important hydrogen bonds and sidechains to the hydrophobic core, and disproportionately contribute to the stability of the domain. However, this stability core is not to be confused with a folding nucleus, which would influence the rate of folding [22,33,51,54,63]. Of course, hydrogen bonds are electrostatic interactions and it is reasonable that those in a low dielectric region of the protein (in the hydrophobic core) would contribute more to stability than those in a solvent-exposed high dielectric region. It remains unclear what subset of molecular information is necessary and sufficient to achieve a b-sheet fold. However, from the perspective of stability alone (not yet considering the pathways by which the fold is acquired, vide infra), it is clear that only a subset of the dense network of interatomic interactions observed in protein structures is required [22,23,33,51–53,54,63,64,65]. This suggests that proteins could maintain their native state even after extensive sidechain and backbone mutagenesis, as long as the critical subset of interactions was maintained. This observation, if it proves to be general, which we expect to be the case, could enable proteins to be substantially modified without compromising structure or function for applications including pharmaceutical uses. Current Opinion in Structural Biology 2006, 16:94–101
96 Folding and binding
Figure 2
Structure of the PIN WW domain. (a) Cartoon of the PIN WW domain structure, indicating residues important for the TS structure and energetics (and therefore the folding rate) in red, and residues important for native state thermodynamic stability in blue. Arrows indicate hydrogen bonds, with the arrowheads pointing towards the acceptor. (b) Structure of the PIN WW domain backbone, with the residues, strands and loops labeled. Residues with sidechains in the hydrophobic core are identified as blue ovals. Buried hydrogen bonds are shown in red, whereas solvent-exposed hydrogen bonds are shown in blue. (c) PIN WW domain structure, with the surface of the hydrophobic core shown in transparent blue and the energetically important hydrogen bonds shown in red. Adapted from [54].
As stated above, the folding funnel hypothesis implies that the acquisition of the native state could occur by many pathways in cases in which the folding funnel is relatively smooth (Figure 1a), making it very difficult to
understand the structural requirements for the nucleation of b-sheet folding [46]. By contrast, roughness in the energy landscape (i.e. energetic barriers substantially larger than kT between individual conformations) would
Figure 3
The effect of A-to-E and A-to-O mutations on hydrogen bonding. An A-to-E mutation weakens one hydrogen bond (as the ester carbonyl is a weaker hydrogen-bond acceptor than an amide carbonyl) and eliminates one hydrogen bond (because the non-carbonyl ester oxygen cannot donate a hydrogen bond). The eliminated hydrogen bond is replaced by modest electrostatic repulsion between the non-carbonyl ester oxygen and the adjacent amide carbonyl. An A-to-O mutation simply removes two hydrogen bonds. Current Opinion in Structural Biology 2006, 16:94–101
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bias the routes taken by most proteins down the folding funnel, such that a family of related pathways or mechanisms would lead to a given b-sheet fold (Figure 1b) [45]. The experimental data collected suggest that b-sheet landscapes are generally rough enough to extract structural information about the transition state (TS) from energetic analyses of mutants [66]. Although it is important to begin to think about the collection and interpretation of protein folding kinetic and thermodynamic data from the perspective of multiple folding pathways for a given sequence, this has yet to be widely embraced by experimentalists.
Kinetic mechanism of WW domain folding The differential influence of a sidechain or backbone mutation on the TS and ground state structures can be discerned using the linear free energy analysis popularized by Fersht [22,23,33,51–53,54,66]. The so-called Fvalue analysis allows one to interrogate the TS structure: FM ¼
y y DDGyf DGf;mut DGf;wt ¼ DDGf DGf;mut DGf;wt
where DGf,mut and DGf,wt are folding free energies, and DDGyf;mut and DDGyf;wt are the activation free energies (i.e.
the free energy difference between the TS and the unfolded state) of the mutant and wild-type proteins, respectively. Values of DDGyf can be extracted from measurements of the folding kinetics. The relaxation time constants exhibited by WW domains are in the 5– 200 ms range (rate constants of 104–105 s1), requiring the use of the laser T-jump method to interrogate their rapid folding [22,23,33,51–53,54]. A FM value of 1 indicates that the interactions perturbed by a mutation stabilize or destabilize the folding TS and the native state to the same extent, that is, the protein has native-like structure in the vicinity of the mutation in the TS [66]. A FM value of 0 indicates the opposite scenario; the protein is unstructured in the vicinity of the mutation in the folding TS. Intermediate values of FM are more difficult to interpret [67], but for proteins exhibiting minimal frustration in their folding landscapes, intermediate FM values represent the average extent to which perturbed interactions exist in the folding TS. Extensive traditional mutagenesis of WW domains reveals FM values of 0.2–0.6 for all secondary structure regions except loop 1, which exhibits high values (FM > 0.8), indicating the formation of loop 1 in the TS [22,33,52,53] (Figure 4a). FM values exceeding 1 are
Figure 4
Summary of PIN WW domain FM values for (a) traditional sidechain variants and (b) A-to-E mutations. Residues for which FM values have been measured are shown in bold. www.sciencedirect.com
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98 Folding and binding
generally interpreted as being due to non-native interactions in the TS [68] that become non-existent at higher temperature. The FM values resulting from A-to-E backbone mutations of the WW domain are particularly informative because, unlike traditional sidechain mutations, they directly describe the hydrogen-bond-mediated formation of secondary structure in the TS in the region proximal to the mutation [54,63]. Data from an A-to-E scan of the WW domain backbone reveal that loop 1 is structured, but not yet native like in the folding TS (FM values approaching 1), consistent with the sidechain perturbation data [54] (Figure 4b). Significant progress by several laboratories studying the mechanisms of bsheet folding reveals that turn formation is often the ratelimiting step in b-sheet and b-hairpin formation, although under certain conditions hydrophobic collapse can become rate limiting [22,23,33,54,69–71,72,73,74, 75,76]. For example, the TS of WW domains can shift appreciably as the folding temperature is varied from 37 8C [33,54,75]. The high FM values exhibited by loop 1 of PIN WW domains (Figure 4) imply that WW domain folding could become faster if the six-residue loop with an internal bturn was transformed into a five- or four-residue turn with a sequence optimized to reverse the direction of the polypeptide chain while maintaining the right-handed twist found in the majority of WW domains. Grafting optimal turn sequences into a PIN host sequence in lieu of loop 1 affords the fastest folding WW domains characterized to date, exhibiting folding time constants of 5 ms, within an order of magnitude of the time constants exhibited by b-hairpins [23]. Interestingly, grafting these fast folding shorter sequences reveals that loop 1 now also substantially influences thermodynamic stability. This effort demonstrates that loop engineering can lower the TS energy by more than 1 kcal/mol (as determined from a two-state Kramers analysis), transforming the WW domain into a near-downhill folder, that is, a sequence that folds into a b-sheet with barriers <0.5 kcal/mol and therefore exhibits a relatively smooth folding funnel (Figure 1a). The evolutionary record of the 120 WW domain family members can be mined to understand the requirements for structure and function [64,65]. Ranganathan and colleagues interrogated each position of the WW domain family with regard to the deviation of the amino acid distribution at that position from the mean distribution found in all proteins, allowing the identification of conserved residues [64,65]. They next identified residue positions exhibiting co-evolution. It is interesting to compare those residues deemed to be important from a co-evolution perspective with those analogously classified as being important on energetic grounds. With regard to fold stability, only two residues, Phe25 and Asn26 (numbering as in this review), have been flagged as important Current Opinion in Structural Biology 2006, 16:94–101
by both perturbation energetic measurements (mutagenesis) and Ranganathan’s statistical coupling analysis [64,65]. Totally or very highly conserved positions, such as Trp11, Tyr23, Trp34 and Pro37, must be formally treated as having the potential for co-evolution, but the lack of variation at these positions precludes the analysis used by Ranganathan. In the case of the two invariant residues for which these domains are named, previous mutagenesis data demonstrate that Trp11 appears to be crucial for thermodynamic stability, whereas Trp34 appears to be important for function [49]. Mutagenesis of residues Glu12, His27 and Ser32, identified as being important from a co-evolution perspective, only modestly alters energetics, indicating that these residues are probably important for WW domain function or dynamics, but do not principally influence folding energetics. The statistical coupling analysis matrix for assessing the co-evolution of residues is very powerful in that it allows hypotheses about the evolutionary constraints on a protein to be tested experimentally. The energetics of folding and function may manifest as statistical couplings, and it will be interesting to discern from future studies whether these and other features are distinct, overlapping or inseparable features of the total sequence information. The coevolution data, combined with Ranganathan’s ability to computationally design WW domains that fold and function by retaining conserved and co-evolving residues only (no tertiary structure information is used), indicate that a relatively small subset of the molecular interactions observed in WW domain structures may be sufficient for folding and function [64,65]. It remains to be discerned whether this is the case for other b-sheet folds and proteins in general, although we expect that, in the case of larger proteins, this small set of interactions could be distributed throughout the sequence. The advantage of studying the evolutionary record without being biased by structural information is that it has the potential to reveal critical residues in b-sheets that perform functions in proteins that we do not currently understand.
Conclusions The combined use of A-to-E and sidechain mutagenesis reinforces the take-home message that loop or reverse turn formation is rate limiting in b-sheet folding. The data obtained from these perturbations also highlights that only a subset of the molecular interactions observed in a protein structure, namely those comprising the hydrophobic core and facilitating buried hydrogen-bond formation, contributes significantly to the thermodynamics of b-sheet formation. This information, along with information mined from the evolutionary record, offers the potential to design proteins with primary structures that only modestly resemble those of naturally occurring proteins, but are nevertheless folded and functional. www.sciencedirect.com
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This paper demonstrates that shorter turns, which reduce the entropic penalty for loop formation, afford the fastest folding hairpins. For threestranded sheets, this is also generally true; however, five-residue turn sequences often fold faster than b-turns (see [23]). 75. Dyer RB, Maness SJ, Franzen S, Fesinmeyer RM, Olsen KA, Andersen NH: Hairpin folding dynamics: the cold-denatured state is predisposed for rapid refolding. Biochemistry 2005, 44:10406-10415. 76. Chen RPY, Huang JJT, Chen HL, Jan H, Velusamy M, Lee CT, Fann WS, Larsen RW, Chan SI: Measuring the refolding of beta-sheets with different turn sequences on a nanosecond time scale. Proc Natl Acad Sci USA 2004, 101:7305-7310. A photolabile cage strategy was used in combination with laser-flash photolysis and photoacoustic calorimetry to demonstrate that turn formation is rate limiting in hairpin formation. Even very subtle changes in sequence can substantially alter folding rates.
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