Role of Side-chains in the Cooperative β-Hairpin Folding of the Short C−Terminal Fragment Derived from Streptococcal Protein G

202

287

Both turn sequence and interstrand hydrophobic side-chain-sidechain interaction have been suggested to be important determinants of ␤-hairpin stability. However, their roles in controlling the folding dynamics of ␤-hairpins have not been clearly determined. Herein, we investigated the structural stability and folding kinetics of a series of tryptophan zippers by static IR and CD spectroscopies and the IR temperature jump method. Our results support a ␤-hairpin folding mechanism wherein the rate-limiting event corresponds to the formation of the turn. We find that the logarithm of the folding rate depends linearly on the entropic change associated with the turn formation, where faster folding correlates with lower entropic cost. Moreover, a stronger turn-promoting sequence increases the stability of a ␤-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a ␤-hairpin primarily by decreasing its unfolding rate.S mall size and structural simplicity make short peptides that fold into well defined structures ideal model systems for examining factors that govern protein folding (1). Of particular interest are ␤-hairpins. With two antiparallel ␤-strands connected by a turn (or loop), the ␤-hairpin motif may be regarded as the smallest folding unit that contains tertiary contacts. Although an increasing body of evidence suggests that the ␤-hairpin can act as a folding nucleus (2-4), the mechanism by which individual ␤-hairpins fold has remained elusive. This elusiveness is partly due to the fact that so far only the folding kinetics of a few sequence-unrelated ␤-hairpins have been studied experimentally (5-9). These studies firmly demonstrated that ␤-hairpins fold on the microsecond time scale; however, the marked difference in the peptide sequence of those systems studied makes it difficult to determine explicitly the key factors that control the rate of ␤-hairpin folding.Although experimental measurements of the folding kinetics of ␤-hairpins are scarce, in the past few years a remarkable number of theoretical and computational studies have been conducted regarding the folding dynamics and energetics of a variety of ␤-hairpin systems (10-21). Results from these studies generally support the idea that the peptide sequence is an important determinant of the folding rate of ␤-hairpins. For example, the statistical model of Muñoz et al. (10) predicts that moving the hydrophobic cluster one residue closer to the turn will speed up the folding rate by 4 times, whereas the results of Thirumalai and Klimov (14,17) suggest that the turn rigidity plays a rather important role in determining the rate as well as the cooperativity of ␤-hairpin folding. Although it is still under debate whether ␤-hairpin folding begins with the formation of the turn (5, 10), interstrand hydrogen bond (16), or hydrophobic collapse (11), results from simulations generally support the idea that it involves multiple kinetic events, whereas the rate-limiting step may correspond to the assem...

Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Understanding the key factors that control the rate of β-hairpin folding

Zhu

Huang

et al. 2004

202

287

“…The native hydrophobic cluster contains two chemical shift probes of folding. Kobayashi and coworkers (23,(28)(29)(30) have long recognized the value of the Tyr-5-H␦ and Val-14-H␥ up resonance shifts for measuring the folding equilibrium of GB1 hairpin analogs. In our recent examination of more stable analogs this continued to be the case.…”

Section: Resultsmentioning

confidence: 99%

Hairpin folding rates reflect mutations within and remote from the turn region

Olsen

Fesinmeyer

Stewart

et al. 2005

149

Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin (k F 278 ‫؍‬ 1.5 ؋ 10 4 ͞s). GB1 hairpin analogs spanning a wide range of thermodynamic stabilities (⌬G U 298 ‫؍‬ ؊3.09 to ؉3.25 kJ͞mol) were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive sidechain͞side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by ''zipper'' folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.␤-hairpin ͉ exchange broadening ͉ folding dynamics ͉ loop search P rotein engineering experiments indicate that ␤-hairpins appear as transition-state features in numerous folding pathways. Hairpin redesign has resulted in changes in protein-folding mechanisms (1), and hairpin stabilization can accelerate (1-3) or retard (2, 4) protein folding. The protein-folding problem continues to be of high interest for at least three reasons: predicting structures from genome-derived sequences, improving a priori protein-fold design, and enhancing our understanding of the mechanisms of protein misfolding diseases (6, 7). Folding rates have been of particular interest, with more examples of redesigned proteins that fold near the calculated protein-folding speed limit (8) appearing regularly. For ␤-sheet proteins and ␤ oligomers (as found in amyloid fibrils formed from misfolded protein states), hairpin dynamics play a key role.Although there is an increasing body of data on hairpin folding dynamics (9-15), with one exception these have not included a set of probing mutations to address specific questions. That exception (14) suggests that loops with a greater turn preference accelerate folding to a much greater extent than the optimization of hydrophobic interactions in the folded state. Other data (12) suggest that the length of the loop connecting the hydrophobic residues that form hairpin-stabilizing cross-strand interactions is reflected in the folding rate. Throughout, hairpin folding has been modeled as a two-state equilibrium. Whether hairpin͞coil transitions are 1-s versus 50-s events has significant consequences. Peptide helix nucleation is a sub-s event (10,16,17), which allows helix formation to be a preequilibrium event relative to the hydrophobic-collapse stage o...

“…We present results for the folding of the model system corresponding to the C-terminal peptide from the B1 domain of protein G (the G peptide), which is known to form a ␤-hairpin structure in solution (34) and has been the subject of previous experimental (35)(36)(37) and computational (7, 8, 12-17, 20, 21, 30, 32, 33, 38) studies. Two mechanisms for G-peptide formation have been previously proposed: the zipper model (35) and the hydrophobic collapse model (20,36,37).…”

mentioning

confidence: 99%

“…Two mechanisms for G-peptide formation have been previously proposed: the zipper model (35) and the hydrophobic collapse model (20,36,37). These models describe the sequence of ␤-hairpin hydrogen bond formation and association of hydrophobic core residues.…”

mentioning

confidence: 99%

Protein folding pathways from replica exchange simulations and a kinetic network model

Andrec

Felts

Gallicchio

et al. 2005

138

167

We present an approach to the study of protein folding that uses the combined power of replica exchange simulations and a network model for the kinetics. We carry out replica exchange simulations to generate a large (Ϸ10 6 ) set of states with an all-atom effective potential function and construct a kinetic model for folding, using an ansatz that allows kinetic transitions between states based on structural similarity. We use this network to perform random walks in the state space and examine the overall network structure. Results are presented for the C-terminal peptide from the B1 domain of protein G. The kinetics is two-state after small temperature perturbations. However, the coil-to-hairpin folding is dominated by pathways that visit metastable helical conformations. We propose possible mechanisms for the ␣-helix͞ ␤-hairpin interconversion.graph ͉ master equation ͉ protein G P rotein folding is a fundamental problem in modern structural biology, and recent advances in experimental techniques have helped to elucidate some of the thermodynamic and kinetic features that underlie different stages of the folding process (1-3). Computer simulations using reduced (4-11) and atomistic molecular models (12-21) have played a central role in the interpretation of experimental studies. Although reduced models have provided considerable insight into the overall mechanisms of protein folding, especially in helping to clarify the nature of folding funnels, intermediates, and kinetic bottlenecks, they are limited in their ability to explore the detailed molecular aspects of folding. However, because of the large number of degrees of freedom and the rarity of folding events even in small model systems, all-atom simulations require both considerable computational resources and ingenuity to obtain meaningful results; a variety of methods have been developed to increase simulation efficiency.A particularly powerful technique for the calculation of free energy surfaces and other thermodynamic properties of complex systems is replica exchange molecular dynamics (REMD) (22), in which a generalized ensemble of the system is simulated in parallel over a range of temperatures. Periodically, coordinates are exchanged by using a Metropolis criterion that ensures that at any given temperature a canonical distribution is realized and allows for more efficient barrier crossing than could be obtained with conventional simulation methods. Although REMD is a powerful method for exploring free energy landscapes, it does not provide direct information about kinetics. To study folding by using all-atom effective potentials, heterogeneous distributed computing (16,19) and transition path sampling (23, 24) techniques have been used. Although the former can enhance sampling by combining information from a large number of short MD trajectories ''steered'' by rare events, these techniques can introduce a bias toward fast events in the ensemble average of the reactive trajectories (25). Transition path sampling (23,24) can yield quantitative estimat...