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.     

Mechanical unfolding of a beta-hairpin using molecular dynamics

Single-molecule mechanical unfolding experiments have the potential to provide insights into the details of protein folding pathways. To investigate the relationship between force-extension unfolding curves and microscopic events, we performed molecular dynamics simulations of the mechanical unfolding of the C-terminal hairpin of protein G. We have studied the dependence of the unfolding pathway on pulling speed, cantilever stiffness, and attachment points. Under conditions that generate low forces, the unfolding trajectory mimics the untethered, thermally accessible pathway previously proposed based on high-temperature studies. In this stepwise pathway, complete breakdown of backbone hydrogen bonds precedes dissociation of the hydrophobic cluster. Under more extreme conditions, the cluster and hydrogen bonds break simultaneously. Transitions between folding intermediates can be identified in our simulations as features of the calculated force-extension curves.     

Molecular dynamics simulations of a beta-hairpin fragment of protein G: balance between side-chain and backbone forces

How is the native structure encoded in the amino acid sequence? For the traditional backbone centric view, the dominant forces are hydrogen bonds (backbone) and phi-psi propensity. The role of hydrophobicity is non-specific. For the side-chain centric view, the dominant force of protein folding is hydrophobicity. In order to understand the balance between backbone and side-chain forces, we have studied the contributions of three components of a beta-hairpin peptide: turn, backbone hydrogen bonding and side-chain interactions, of a 16-residue fragment of protein G. The peptide folds rapidly and cooperatively to a conformation with a defined secondary structure and a packed hydrophobic cluster of aromatic side-chains. Our strategy is to observe the structural stability of the beta-hairpin under systematic perturbations of the turn region, backbone hydrogen bonds and the hydrophobic core formed by the side-chains, respectively. In our molecular dynamics simulations, the peptides are solvated. with explicit water molecules, and an all-atom force field (CFF91) is used. Starting from the original peptide (G41EWTYDDATKTFTVTE56), we carried out the following MD simulations. (1) unfolding at 350 K; (2) forcing the distance between the C(alpha) atoms of ASP47 and LYS50 to be 8 A; (3) deleting two turn residues (Ala48 and Thr49) to form a beta-sheet complex of two short peptides, GEWTYDD and KTFTVTE; (4) four hydrophobic residues (W43, Y45, F52 and T53) are replaced by a glycine residue step-by-step; and (5) most importantly, four amide hydrogen atoms (T44, D46, T53, and T55, which are crucial for backbone hydrogen bonding), are substituted by fluorine atoms. The fluorination not only makes it impossible to form attractive hydrogen bonding between the two beta-hairpin strands, but also introduces a repulsive force between the two strands due to the negative charges on the fluorine and oxygen atoms. Throughout all simulations, we observe that backbone hydrogen bonds are very sensitive to the perturbations and are easily broken. In contrast, the hydrophobic core survives most perturbations. In the decisive test of fluorination, the fluorinated peptide remains folded under our simulation conditions (5 ns, 278 K). Hydrophobic interactions keep the peptide folded, even with a repulsive force between the beta-strands. Thus, our results strongly support a side-chain centric view for protein folding.     

A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK

Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate(s) were generally small (<25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of N-PGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems.     

Understanding the mechanism of beta-sheet folding from a chemical and biological perspective

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.     

Beta-sheet folding mechanisms from perturbation energetics

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 beta-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 beta-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 beta-sheets, consistent with evolutionary robustness.     

Specific non-native hydrophobic interactions in a hidden folding intermediate: implications for protein folding

Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates.     

Ultrafast folding and molecular dynamics of a linear hydrophobic β-hairpin

The first computational study of the folding and dynamics of a hydrophobic β-hairpin containing a central heterochiral diproline segment is reported. Linear hydrophobic sequences containing centrally positioned diproline motifs, heterochiral (DL/LD) and homochiral (LL/DD)), are investigated for their ability to form β-hairpins. Heterochiral diproline motifs (LD/DL) reveal the formation of stable β-hairpins with the backbone adopting β-turn conformation and the formation of backbone hydrogen bonds with antiparallel cross-strand registry, whereas the homochiral diproline (LL/DD) containing sequences tend to adopt PP_II helix conformation. The competition between the β-turn formation and the backbone H-bond ladder of the antiparallel β-strands in heterochiral diproline containing sequences is employed to validate the hypothesis that β-turn formation precedes inter-strand registry in the folding of a β-hairpin (“zipper” mechanism). The observation of noncanonical hydrogen bonds leads to a folded β-hairpin-like conformation and points to the existence of relatively stable transition state intermediates, between the unfolded (extended) and folded (β-hairpin) states. The MD simulations are in excellent agreement with the experimental studies on the model system and constitute the very first computational investigation of the folding and dynamics of a completely hydrophobic synthetic β-hairpin containing heterogeneous residues of mixed chirality.     

Folding of a DNA hairpin loop structure in explicit solvent using replica-exchange molecular dynamics simulations

Hairpin loop structures are common motifs in folded nucleic acids. The 5'-GCGCAGC sequence in DNA forms a characteristic and stable trinucleotide hairpin loop flanked by a two basepair stem helix. To better understand the structure formation of this hairpin loop motif in atomic detail, we employed replica-exchange molecular dynamics (RexMD) simulations starting from a single-stranded DNA conformation. In two independent 36 ns RexMD simulations, conformations in very close agreement with the experimental hairpin structure were sampled as dominant conformations (lowest free energy state) during the final phase of the RexMDs ( approximately 35% at the lowest temperature replica). Simultaneous compaction and accumulation of folded structures were observed. Comparison of the GCA trinucleotides from early stages of the simulations with the folded topology indicated a variety of central loop conformations, but arrangements close to experiment that are sampled before the fully folded structure also appeared. Most of these intermediates included a stacking of the C(2) and G(3) bases, which was further stabilized by hydrogen bonding to the A(5) base and a strongly bound water molecule bridging the C(2) and A(5) in the DNA minor groove. The simulations suggest a folding mechanism where these intermediates can rapidly proceed toward the fully folded hairpin and emphasize the importance of loop and stem nucleotide interactions for hairpin folding. In one simulation, a loop motif with G(3) in syn conformation (dihedral flip at N-glycosidic bond) accumulated, resulting in a misfolded hairpin. Such conformations may correspond to long-lived trapped states that have been postulated to account for the folding kinetics of nucleic acid hairpins that are slower than expected for a semiflexible polymer of the same size.     

High temperature unfolding simulations of the TRPZ1 peptide

We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 μs with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.     

Effects of Turn Stability and Side-Chain Hydrophobicity on the Folding of β-Structures

Key elements of β-structure folding include hydrophobic core collapse, turn formation, and assembly of backbone hydrogen bonds. In the present folding simulations of several β-hairpins and β-sheets (peptide 1, protein G B1 domain peptide, TRPZIP2, TRPZIP4, 20mer, and 20mer^DP6D), the folding free-energy landscape as a function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on turn stability and side-chain hydrophobicity, which demonstrates different folding mechanisms of similar β-structures of varied sequences. Turn stability is found to be the key factor in determining the formation order of the three structural elements in the folding of β-structures. Moreover, turn stability and side-chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded β-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stabilities of two anti-parallel hairpins in each three-stranded β-sheet are shown to correlate well with the different levels of their hydrophobic interactions.     

The chicken-egg scenario of protein folding revisited

What is the first step in protein folding - hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non-compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding.     

Direct analysis of backbone-backbone hydrogen bond formation in protein folding transition states

Here we investigate the role of backbone-backbone hydrogen bonding interactions in stabilizing the protein folding transition states of two model protein systems, the B1 domain of protein L (ProtL) and the P22 Arc repressor. A backbone modified analogue of ProtL containing an amide-to-ester bond substitution between residues 105 and 106 was prepared by total chemical synthesis, and the thermodynamic and kinetic parameters associated with its folding reaction were evaluated. Ultimately, these parameters were used in a Phi-value analysis to determine if the native backbone-backbone hydrogen bonding interaction perturbed in this analogue (i.e. a hydrogen bond in the first beta-turn of ProtL's beta-beta-alpha-beta-beta fold) was formed in the transition state of ProtL's folding reaction. Also determined were the kinetic parameters associated with the folding reactions of two Arc repressor analogues, each containing an amide-to-ester bond substitution in the backbone of their polypeptide chains. These parameters were used together with previously established thermodynamic parameters for the folding of these analogues in Phi-value analyses to determine if the native backbone-backbone hydrogen bonding interactions perturbed in these analogues (i.e. a hydrogen bond at the end of the intersubunit beta-sheet interface and hydrogen bonds at the beginning of the second alpha-helix in Arc repressor's beta-alpha-alpha structure) were formed in the transition state of Arc repressor's folding reaction. Our results reveal that backbone-backbone hydrogen bonding interactions are formed in the beta-turn and alpha-helical transition state structures of ProtL and Arc repressor, respectively; and they were not formed in the intersubunit beta-sheet interface of Arc repressor, a region of Arc repressor's polypeptide chain previously shown to have other non-native-like conformations in Arc's protein folding transition state.     

Inversion of the balance between hydrophobic and hydrogen bonding interactions in protein folding and aggregation

Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.     

Kinetic pathways of beta-hairpin (un)folding in explicit solvent

We examine the dynamical (un)folding pathways of the C-terminal beta-hairpin of protein G-B1 at room temperature in explicit solvent, by employing transition path sampling algorithms. The path ensembles contain information on the folding kinetics, including solvent motion. We determine the transition state ensembles for the two main transitions: 1), the hydrophobic collapse; and 2), the backbone hydrogen bond formation. In both cases the transition state ensembles are characterized by a layer (1) or a strip (2) of water molecules in between the two hairpin strands, supporting the hypothesis of the solvent as lubricant in the folding process. The transition state ensembles do not correspond with saddle points in the equilibrium free-energy landscapes. The kinetic pathways are thus not completely determined by the free-energy landscape. This phenomenon can occur if the order parameters obey different timescales. Using the transition interface sampling technique, we calculate the rate constants for (un)folding and find them in reasonable agreement with experiments, thus supporting the validation of using all-atom force fields to study protein folding.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

(Un)Folding Mechanisms of the FBP28 WW Domain in Explicit Solvent Revealed by Multiple Rare Event Simulation Methods

We report a numerical study of the (un)folding routes of the truncated FBP28 WW domain at ambient conditions using a combination of four advanced rare event molecular simulation techniques. We explore the free energy landscape of the native state, the unfolded state, and possible intermediates, with replica exchange molecular dynamics. Subsequent application of bias-exchange metadynamics yields three tentative unfolding pathways at room temperature. Using these paths to initiate a transition path sampling simulation reveals the existence of two major folding routes, differing in the formation order of the two main hairpins, and in hydrophobic side-chain interactions. Having established that the hairpin strand separation distances can act as reasonable reaction coordinates, we employ metadynamics to compute the unfolding barriers and find that the barrier with the lowest free energy corresponds with the most likely pathway found by transition path sampling. The unfolding barrier at 300 K is ∼17 k_BT ≈ 42 kJ/mol, in agreement with the experimental unfolding rate constant. This work shows that combining several powerful simulation techniques provides a more complete understanding of the kinetic mechanism of protein folding.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Molecular dynamics simulation of the SH3 domain aggregation suggests a generic amyloidogenesis mechanism

We use molecular dynamics simulation to study the aggregation of Src SH3 domain proteins. For the case of two proteins, we observe two possible aggregation conformations: the closed form dimer and the open aggregation state. The closed dimer is formed by "domain swapping"-the two proteins exchange their RT-loops. All the hydrophobic residues are buried inside the dimer so proteins cannot further aggregate into elongated amyloid fibrils. We find that the open structure-stabilized by backbone hydrogen bond interactions-packs the RT-loops together by swapping the two strands of the RT-loop. The packed RT-loops form a beta-sheet structure and expose the backbone to promote further aggregation. We also simulate more than two proteins, and find that the aggregate adopts a fibrillar double beta-sheet structure, which is formed by packing the RT-loops from different proteins. Our simulations are consistent with a possible generic amyloidogenesis scenario.     

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.