Effect of proline residues on protein folding

Conformational energy calculations have been used to study the role of the proline residues in the folding of bovine pancreatic trypsin inhibitor. In the calculation, each of the four proline residues of this small protein is forced from the trans to cis peptide isomer while still part of the native folded structure. The cis proline residue can always be accommodated by small changes of the native conformation (< 1 Å root-mean-square deviation). For three of the four proline residues, Pro2, Pro9 and Pro 13, being in the cis form is calculated to destabilize the folded conformation by less than 11 kcal/mol, suggesting that rapid folding to a stable native-like conformation can occur with either isomeric form. For one of these three, Pro13, the destabilization is only 1 kcal/mol, suggesting the existence of an alternative folded native conformation with Pro13 cis. The fourth proline residue, Pro8, is calculated to destabilize the native conformation by so much (33 kcal/mol) that it will block folding in the manner proposed by Brandts et al. (1975).     

Impact of proline residues on parvalbumin stability

Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.     

Role of proline residues in the folding of serine hydroxymethyltransferase

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism. The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity. The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered. However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme. Mutation of both Pro258 and Pro264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity. The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability. Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme. The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5'-phosphate addition to the apo-enzyme.     

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.     

Helix mutations stabilize a late productive intermediate on the folding pathway of ubiquitin

We have investigated the relative placement of rate-limiting energy barriers and the role of productive or obstructive intermediates on the folding pathway of yeast wild-type ubiquitin ( wt-Ub) containing the F45W mutation. To manipulate the folding barriers, we have designed a family of mutants in which stabilizing substitutions have been introduced incrementally on the solvent-exposed surface of the main alpha-helix (residues 23-34), which has a low intrinsic helical propensity in the native sequence. Although the U --> I and I --> N transitions are not clearly delineated in the kinetics of wt-Ub, we show that an intermediate becomes highly populated and more clearly resolved as the predicted stability of the helix increases. The observed acceleration in the rate of folding correlates with helix stability and is consistent with the I-state representing a productive rather than misfolded state. A Leffler analysis of the effects on kinetics of changes in stability within the family of helix mutants results in a biphasic correlation in both the refolding and unfolding rates that suggest a shift from a nucleation-condensation mechanism (weakly stabilized helix) toward a diffusion-collision model (highly stabilized helix). Through the introduction of helix-stabilizing mutations, we are able to engineer a well-resolved I-state on the folding pathway of ubiquitin which is likely to be structurally distinct from that which is only weakly populated on the folding pathway of wild-type ubiquitin.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.     

The folding pathway of ubiquitin from all-atom molecular dynamics simulations

The folding (unfolding) pathway of ubiquitin is probed using all-atom molecular dynamics simulations. We dissect the folding pathway using two techniques: first, we probe the folding pathway of ubiquitin by calculating the evolution of structural properties over time and second, we identify the rate determining transition state for folding. The structural properties that we look at are hydrophobic solvent accessible surface area (SASA) and Calpha-root-mean-square deviation (rmsd). These properties on their own tell us relatively little about the folding pathway of ubiquitin; however, when plotted against each other, they become powerful tools for dissecting ubiquitin's folding mechanism. Plots of Calpha-rmsd against SASA serve as a phase space trajectories for the folding of ubiquitin. In this study, these plots show that ubiquitin folds to the native state via the population of an intermediate state. This is shown by an initial hydrophobic collapse phase followed by a second phase of secondary structure arrangement. Analysis of the structure of the intermediate state shows that it is a collapsed species with very little secondary structure. In reconciling these observations with recent experimental data, the transition that we observe in our simulations from the unfolded state (U) to the intermediate state (I) most likely occurs in the dead-time of the stopped flow instrument. The folding pathway of ubiquitin is probed further by identification of the rate-determining transition state for folding. The method used for this is essential dynamics, which utilizes a principal component analysis (PCA) on the atomic fluctuations throughout the simulation. The five transition state structures identified in silico are in good agreement with the experimentally determined transition state. The calculation of phi-values from the structures generated in the simulations is also carried out and it shows a good correlation with the experimentally measured values. An initial analysis of the denatured state shows that it is compact with fluctuating regions of nonnative secondary structure. It is found that the compactness in the denatured state is due to the burial of some hydrophobic residues. We conclude by looking at a correlation between folding kinetics and residual structure in the denatured state. A hierarchical folding mechanism is then proposed for ubiquitin.     

Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin

Background

Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C^γ-exo or a C^γ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C^γ-exo puckering.

Methodology/Principal Findings

While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C^γ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol⁻¹ in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.

Conclusions/Significance

The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Thermodynamic effects of proline introduction on protein stability

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.     

Insights into the stability of native and partially folded states of ubiquitin: effects of cosolvents and denaturants on the thermodynamics of protein folding

The thermodynamics of the native<-->A state and native<-->unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn.HCl) both separately and in combination. Gdn.HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native<-->unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn.HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same "native" state; however, the thermodynamics of the N<-->U transition change dramatically between alcoholic and Gdn.HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in DeltaC(p) at higher methanol concentrations, as previously reported for the N<-->A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P. A. (1993) J. Mol. Biol. 229, 502-511.). Both the N<-->U and the N<-->A transitions are enthalpy driven to a similar extent. We conclude that under these conditions van der Waals interactions in the packing of the nonpolar protein core, which is common to both the N<-->U and the N<-->A transitions, appear to drive folding in the absence of entropic effects associated with release of ordered solvent (hydrophobic effect). Solvent transfer studies of hydrocarbons into alcoholic solvents, with and without Gdn.HCl, are consistent with a large enthalpic driving force for burial of a nonpolar surface, with a linear dependence of protein stability (DeltaG(N)(<-->)(U)) on cosolvent concentration reflected in a similar linear dependence of hydrocarbon solubility. The data demonstrate that the hydrophobic effect is not a prerequisite for specific stabilization of the native state or the A state and that van der Waals packing of the nonpolar core appears to be the dominant factor in stabilization of the native state.     

The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding.

The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter phi. There are two extreme values of phi that are often found in practice and may be interpreted in a simple manner. A value of phi = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, phi = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of phi are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of phi found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of phi = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the double-mutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.     

Identification of proline residues responsible for the slow folding kinetics in pectate lyase C by mutagenesis

The folding mechanism of pectate lyase C (pelC) involves two slow phases that have been attributed to proline isomerization. To have a more detailed and complete understanding of the folding mechanism, experiments have been carried out to identify the prolyl-peptide bonds responsible for the slow kinetics. Site-directed mutagenesis has been used to mutate each of the prolines in pelC to alanine or valine. It has been determined that isomerization of the Leu219-Pro220 peptide bond is responsible for the slowest folding phase observed. The mutant P220A shows kinetic behavior that is identical to the wild-type protein except that the 46-s phase is eliminated. The Leu219-Pro220 peptide bond is cis in the native enzyme. An analysis of the free energy of unfolding of this mutant indicates that the mutation destabilizes the protein by about 4 kcal/mol. However, it appears that the major refolding pathways are unaltered. Further mutations were carried out in order to assign the peptide bond responsible for the 21-s folding phase in pelC. Mutation of the remaining 11 prolines, which are trans in the native enzyme, resulted in no significant changes in the kinetic folding behavior. The conclusion from these experiments is that the 21-s phase involves isomerization of more than one prolyl-peptide bond with similar activation energies.     

Substitutions of proline 76 in yeast iso-1-cytochrome c. Analysis of residues compatible and incompatible with folding requirements

Fine-structure genetic mapping previously revealed numerous nonfunctional cyc1 mutations having alterations at or near the site corresponding to amino acid position 76 of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. DNA sequencing of the alterations in four of these cyc1 mutations indicated that the normal Pro-76 was replaced by Leu-76. Revertants containing at least partially functional iso-1-cytochromes c were isolated, and the alterations were analyzed by DNA sequencing and protein analysis. Specific activities of the altered iso-1-cytochromes c were estimated in vivo by growth of the strains in lactate medium; compared to normal iso-1-cytochrome c with Pro-76, the following activities were associated with the following replacements: approximately 90% for Val-76, approximately 60% for Thr-76, approximately 30% for Ser-76, approximately 20% for Ile-76, and 0% for Leu-76. In order to develop an understanding of the factors that determine whether or not an altered iso-1-cytochrome c will function, we undertook a theoretical analysis which led to the conclusion that the activity of the proteins was dependent on both short- and long-range interactions. Short-range interactions were estimated from studies on known protein structures which gave the likelihood that various amino acids would be found in a local backbone configuration similar to the native protein; long-range interactions with the rest of the molecule were analyzed by considering the size of the side chain. We believe this approach can be used to analyze a wide variety of mutant proteins.     

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β‐turn occurrence, we engineered two chimerical enzymes where their super secondary β‐loop‐α motifs 2 ((βα)₂) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)₂ motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)₂ motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)₂ of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N‐terminal of loop‐2 and the C‐terminal of α‐helix‐2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E_A). A systematic analysis of DSC data showed a large decrease on the E_A of TcTIM (ΔE_A ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571–579. © 2016 Wiley Periodicals, Inc.     

Bacteria and the ubiquitin pathway

Ubiquitylation participates in a repertoire of reversible post-translational modifications that modulate the function, localization and half-life of proteins by regulating their association with various ubiquitin-binding proteins. In response to pathogen infection, bacterial effectors impact ubiquitin and ubiquitin-like modifications of key proteins in immune and anti-apoptotic signaling cascades. Certain bacteria corrupt the ubiquitylation machinery in order to regulate their virulence factors spatially and temporally or to trigger internalization of bacteria into host cells. Several new examples of how bacterial factors target ubiquitin and ubiquitin-like regulation emphasize the importance of modulating ubiquitin signaling to establish either long-lasting or devastating relationships of bacteria with their hosts.     

Role of proline residues in human lysozyme stability: a scanning calorimetric study combined with X-ray structure analysis of proline mutants.

It has been shown that protein stability can be modulated from site-directed mutations that affect the entropy of protein unfolding [Matthews, B. W., Nicholson, H., & Becktel, W. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6663-6667]. However, the effect of a specific amino acid replacement on stability highly depends on the location of the mutation site and its environment in the protein structure [Yutani, K., Hayashi, S., Sugisaki, Y., & Ogasahara, K. (1991) Proteins Struct., Funct., Genet. 9, 90-98). To clarify the role of specific proline residues in the thermostability of human lysozyme (h-lysozyme), a series of proline mutants were investigated by means of scanning calorimetry and high-resolution X-ray crystallography. The thermodynamic properties of the mutant and wild-type h-lysozymes are compared and discussed on the basis of their three-dimensional structure. h-Lysozyme contains two proline residues at positions 71 and 103. The Pro71----Gly substitution was found to destabilize h-lysozyme by decreasing the entropic contribution of unfolding by about 2 kcal/mol at 68.8 degrees C. This is consistent with the theoretical expectations for such a substitution. However, the same substitution at position 103 (Pro103----Gly) does not affect h-lysozyme stability, and the thermodynamic properties of the P71G/P103G and P71G mutants are essentially the same. Pro71 which is conserved among lysozymes from other species, appears to be important for stability, whereas Pro103, which is not conserved, does not. These differences are explained in terms of residue accessibility to the solvent and crystallographic B-factor, which reflects the amino acid mobility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of proline mutations on the folding of staphylococcal nuclease

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.     

Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C^γ ring pucker preference and the cis– trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple‐stranded antiparallel β‐sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C^γ ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C^γ‐endo ring pucker in the native structure. Pro37 was replaced with C^γ‐exo biased pucker derivatives: (2S,4R)‐4‐hydroxyproline (4R‐Hyp), (2S,4R)‐4‐fluoroproline (4R‐Flp), (2S,4R)‐4‐methoxyproline (4R‐Mop), and C^γ‐endo biased pucker derivatives: (2S,4S)‐4‐hydroxyproline (4S‐hyp), (2S,4S)‐4‐fluoroproline (4S‐flp), (2S,4S)‐4‐methoxyproline (4S‐mop) to examine how a preorganized pucker affects the folding stability and ligand‐binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S‐flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C? F group. Analysis of ligand‐binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S‐flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4‐substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.