Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin

The foldon domain constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4. Its function is to promote folding and trimerization of fibritin. We investigated structure, stability and folding mechanism of the isolated foldon domain. The domain folds into the same trimeric beta-propeller structure as in fibritin and undergoes a two-state unfolding transition from folded trimer to unfolded monomers. The folding kinetics involve several consecutive reactions. Structure formation in the region of the single beta-hairpin of each monomer occurs on the submillisecond timescale. This reaction is followed by two consecutive association steps with rate constants of 1.9(+/-0.5)x10(6)M(-1)s(-1) and 5.4(+/-0.3)x10(6)M(-1)s(-1) at 0.58 M GdmCl, respectively. This is similar to the fastest reported bimolecular association reactions for folding of dimeric proteins. At low concentrations of protein, folding shows apparent third-order kinetics. At high concentrations of protein, the reaction becomes almost independent of protein concentrations with a half-time of about 3 ms, indicating that a first-order folding step from a partially folded trimer to the native protein (k=210 +/- 20 s(-1)) becomes rate-limiting. Our results suggest that all steps on the folding/trimerization pathway of the foldon domain are evolutionarily optimized for rapid and specific initiation of trimer formation during fibritin assembly. The results further show that beta-hairpins allow efficient and rapid protein-protein interactions during folding.     

The folding landscape of an alpha-lytic protease variant reveals the role of a conserved beta-hairpin in the development of kinetic stability

Most secreted bacterial proteases, including alpha-lytic protease (alphaLP), are synthesized with covalently attached pro regions necessary for their folding. The alphaLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the alphaLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although alphaLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved beta-hairpin in the C-terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an alphaLP variant with a more favorable beta-hairpin turn conformation (alphaLP(beta-turn)). Indeed, alphaLP(beta-turn) pro region-catalyzed folding is faster than that for alphaLP. However, instead of accelerating spontaneous folding, alphaLP(beta-turn) actually unfolds more slowly than alphaLP. Our data support a model where the beta-hairpin is formed early, but its packing with a loop in the N-terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of alphaLP(beta-turn), to nearly the same degree as the change between alphaLP and a faster folding homolog. However, alphaLP(beta-turn) has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties.     

Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.     

Thermodynamics of a beta-hairpin structure: evidence for cooperative formation of folding nucleus

To elucidate early nucleation stages in protein folding, multi-probed thermodynamic characterization was applied to the beta-hairpin structural formation of G-peptide, which is a C-terminal fragment of the B1 domain of streptococcal protein G. The segment corresponding to the sequence of G-peptide is believed to act as a nucleus during the folding process of the B1 domain. In spite of the broad thermal transition of G-peptide, nuclear magnetic resonance (NMR) melting measurements combined with our original analytical theory enabled us to obtain the thermodynamic properties of the beta-hairpin formation with considerable accuracy. Additionally, all the thermodynamic properties determined by every NMR probe on both the main-chain and the side-chains were quite similar, and also comparable to the values that were independently determined by calorimetric analysis of G-peptide. These results demonstrate that G-peptide folds cooperatively throughout the molecule. In other words, the formation of the beta-hairpin is interpreted as the fashion of a first-order phase transition between two states without any distinguishable intermediates. This cooperative formation of the short linear peptide consisting of only 16 residues provides insight into not only the first folding events of the B1 domain, but also the general principles of proteins in terms of structural hierarchy, stability and folding mechanism.     

Understanding the mechanism of beta-hairpin folding via phi-value analysis

The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.     

Stabilization of short collagen-like triple helices by protein engineering

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.     

Role of side-chains in the cooperative beta-hairpin folding of the short C-terminal fragment derived from streptococcal protein G

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.     

The role of a beta-bulge in the folding of the beta-hairpin structure in ubiquitin

It is known that the peptide corresponding to the N-terminal beta-hairpin of ubiquitin, U(1-17), can populate the monomeric beta-hairpin conformation in aqueous solution. In this study, we show that the Gly-10 that forms the bulge of the beta-turn in this hairpin is very important to the stability of the hairpin. The deletion of this residue to desG10(1-16) unfolds the structure of the peptide in water. Even under denaturing conditions, this bulge appears to be important in maintaining the residual structure of ubiquitin, which involves tertiary interactions within the sequence 1 to 34 in the denatured state. We surmise that this residual structure functions as one of the nucleation centers in the folding process and is important in stabilizing the transition state. In accordance with this idea, deleting Gly-10 slows down the refolding and unfolding rate by about one half.     

Identification of a key structural element for protein folding within beta-hairpin turns

Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.     

Folding and aggregation kinetics of a beta-hairpin

We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.     

The carboxy-terminal domain initiates trimerization of bacteriophage T4 fibritin.

Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. Earlier we showed that the C-terminal globular domain (foldon) of fibritin is essential for correct trimerization and folding of the protein. We constructed the chimerical fusion protein W31 in which the fibritin foldon sequence is followed by the small globular non-alpha-helical protein gp31 of the T4 phage. We showed that the foldon is capable of trimerization in the absence of the coiled-coil part of fibritin. A deletion mutant of fibritin (NB1) with completely deleted foldon is unable to fold and trimerize correctly. An excess of this mutant protein did not influence the refolding of fibritin in vitro, and the chimerical protein inhibited this process efficiently. Our conclusion is that the trimerization of the foldon is the initial step of fibritin refolding and is followed by the formation of the coiled-coil structure.     

Engineering diverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state

Using the N-terminal 17-residue beta-hairpin of ubiquitin as a "host" for mutational studies, we have investigated the influence of the beta-turn sequence on protein stability and folding kinetics by replacing the native G-bulged turn (TLTGK) with more flexible analogues (TG3K and TG5K) and a series of four-residue type I' beta-turn sequences, commonly found in beta-hairpins. Although a statistical analysis of type I' turns demonstrates residue preferences at specific sites, the frequency of occurrence appears to only broadly correlate with experimentally determined protein stabilities. The subsequent engineering of context-dependent non-native tertiary contacts involving turn residues is shown to produce large changes in stability. Relatively few point mutations have been described that probe secondary structure formation in ubiquitin in a manner that is independent of tertiary contacts. To this end, we have used the more rigorous rate-equilibrium free energy relationship (Leffler analysis), rather than the two-point phi value analysis, to show for a family of engineered beta-turn mutants that stability (range of approximately 20 kJ/mol) and folding kinetics (190-fold variation in refolding rate) are linearly correlated (alpha(f) = 0.74 +/- 0.08). The data are consistent with a transition state that is robust with regard to a wide range of statistically favored and disfavored beta-turn mutations and implicate a loosely assembled beta-hairpin as a key template in transition state stabilization with the beta-turn playing a central role.     

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.     

Understanding beta-hairpin formation by molecular dynamics simulations of unfolding

We have studied the mechanism of formation of a 16-residue beta-hairpin from the protein GB1 using molecular dynamics simulations in an aqueous environment. The analysis of unfolding trajectories at high temperatures suggests a refolding pathway consisting of several transient intermediates. The changes in the interaction energies of residues are related with the structural changes during the unfolding of the hairpin. The electrostatic energies of the residues in the turn region are found to be responsible for the transition between the folded state and the hydrophobic core state. The van der Waals interaction energies of the residues in the hydrophobic core reflect the behavior of the radius of gyration of the core region. We have examined the opposing influences of the protein-protein (PP) energy, which favors the native state, and the protein-solvent (PS) energy, which favors unfolding, in the formation of the beta-hairpin structure. It is found that the behavior of the electrostatic components of PP and PS energies reflects the structural changes associated with the loss of backbone hydrogen bonding. Relative changes in the PP and PS van der Waals interactions are related with the disruption of the hydrophobic core of a protein. The results of the simulations support the hydrophobic collapse mechanism of beta-hairpin folding.     

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

Molecular dynamics simulations of beta-hairpin folding

Molecular dynamics simulations of beta-hairpin folding have been carried out with a solvent-referenced potential at 274 K. The model peptide V4DPGV4 formed stable beta-hairpin conformations and the beta-hairpin ratio calculated by the DSSP algorithm was about 56% in the 50-ns simulation. Folding into beta-hairpin conformations is independent of the initial conformations. The simulations provided insights into the folding mechanism. The hydrogen bond often formed in a beta-turn first, and then propagated by forming more hydrogen bonds along the strands. Unfolding and refolding occurred repeatedly during the simulations. Both the hydrogen bonding and the hydrophobic interaction played important roles in forming the ordered structure. Without the hydrophobic effect, stable beta-hairpin conformations did not form in the simulations. With the same energy functions, the alanine-based peptide (AAQAA)3Y folded into helical conformations, in agreement with experiments. Folding into an alpha-helix or a beta-hairpin is amino acid sequence-dependent.     

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.     

Nanosecond temperature jump relaxation dynamics of cyclic beta-hairpin peptides

The thermal unfolding of a series of 6-, 10-, and 14-mer cyclic beta-hairpin peptides was studied to gain insight into the mechanism of formation of this important secondary structure. The thermodynamics of the transition were characterized using temperature dependent Fourier transform infrared spectroscopy. Thermodynamic data were analyzed using a two-state model which indicates increasing cooperativity along the series. The relaxation kinetics of the peptides in response to a laser induced temperature jump were probed using time-resolved infrared spectroscopy. Single exponential relaxation kinetics were observed and fit with a two-state model. The folding rate determined for these cyclic peptides is accelerated by some two orders of magnitude over the rate of a linear peptide that forms a beta-hairpin. This observation supports the argument that the rate limiting step in the linear system is either stabilization of compact collapsed structures or rearrangement of collapsed structures over a barrier to achieve the native interstrand registry. Small activation energies for folding of these peptides obtained from an Arrhenius analysis of the rates imply a primarily entropic barrier, hence an organized transition state having specific stabilizing interactions.     

The role of the turn in beta-hairpin formation during WW domain folding

The folding of WW domains is rate limited by formation of a beta-hairpin comprising residues from strands 1 and 2. Residues in the turn of this hairpin have reported Phi-values for folding close to 1 and have been proposed to nucleate folding. High Phi-values do not necessarily imply that the energetics of formation are a driving force for initiating folding. We demonstrate by NMR studies and molecular dynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurally dynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that the formation of the beta-turn per se provides the energetic driving force for hairpin folding. It is more likely that the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleus as defined by the nucleation-condensation mechanism whereby a diffuse nucleus is stabilized by associated interactions.