A residual structure in unfolded intestinal fatty acid binding protein consists of amino acids that are neighbors in the native state

Much of the recent effort in protein folding has focused on the possibility that residual structures in the unfolded state may provide an initiating site for protein folding. This hypothesis is difficult to test because of the weak stability and dynamic behavior of these structures. This problem has been simplified for intestinal fatty acid binding protein (IFABP) by incorporating fluorinated aromatic amino acids during synthesis in Escherichia coli. Only the labeled residues give signals by (19)F NMR, and the 1D spectra can be assigned in both the native and unfolded states by site-directed mutagenesis. One of the two tryptophans (W82), one of the four tyrosines (Y70), and at least four of the eight phenylalanines (including F68 and F93) of IFABP are involved in a structure that is significantly populated at concentrations of urea that unfold the native structure by fluorescence and CD criteria. These residues are nonlocal in sequence and also contact each other in the native structure. Thus, a template of nativelike hydrophobic contacts in the unfolded state may serve as an initiating site for folding this beta-sheet protein.     

Cooperative folding mechanism of a beta-hairpin peptide studied by a multicanonical replica-exchange molecular dynamics simulation

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.     

Identifying folding nucleus based on residue contact networks of proteins

In the native structure of a protein, all the residues are tightly parked together in a specific order following its folding and every residue contacts with some spatially neighbor residues. A residue contact network can be constructed by defining the residues as nodes and the native contacts as edges. During the folding of small single-domain proteins, there is a set of contacts (or bonds), defined as the folding nucleus (FN), which is formed around the transition state, i.e., a rate-limiting barrier located at about the middle between the unfolded states and the native state on the free energy landscape. Such a FN plays an essential role in the folding dynamics and the residues, which form the related contacts called as folding nucleus residues (FNRs). In this work, the FNRs in proteins are identified by using quantities which characterize the topology of residue contact networks of proteins. By comparing the specificities of residues with the network quantities K(R), L(R), and D(R), up to 90% FNRs of six typical proteins found experimentally are identified. It is found that the FNRs behave the full-closeness centrals rather than degree or closeness centers in the residue contact network, implying that they are important to the folding cooperativity of proteins. Our study shows that the FNRs can be identified solely from the native structures of proteins based on the analysis of residue contact network without any knowledge of the transition state ensemble.     

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

The Limited Role of Nonnative Contacts in the Folding Pathways of a Lattice Protein

Models of protein energetics that neglect interactions between amino acids that are not adjacent in the native state, such as the Gō model, encode or underlie many influential ideas on protein folding. Implicit in this simplification is a crucial assumption that has never been critically evaluated in a broad context: Detailed mechanisms of protein folding are not biased by nonnative contacts, typically argued to be a consequence of sequence design and/or topology. Here we present, using computer simulations of a well-studied lattice heteropolymer model, the first systematic test of this oft-assumed correspondence over the statistically significant range of hundreds of thousands of amino acid sequences that fold to the same native structure. Contrary to previous conjectures, we find a multiplicity of folding mechanisms, suggesting that Gō-like models cannot be justified by considerations of topology alone. Instead, we find that the crucial factor in discriminating among topological pathways is the heterogeneity of native contact energies: The order in which native contacts accumulate is profoundly insensitive to omission of nonnative interactions, provided that native contact heterogeneity is retained. This robustness holds over a surprisingly wide range of folding rates for our designed sequences. Mirroring predictions based on the principle of minimum frustration, fast-folding sequences match their Gō-like counterparts in both topological mechanism and transit times. Less optimized sequences dwell much longer in the unfolded state and/or off-pathway intermediates than do Gō-like models. For dynamics that bridge unfolded and unfolded states, however, even slow folders exhibit topological mechanisms and transit times nearly identical with those of their Gō-like counterparts. Our results do not imply a direct correspondence between folding trajectories of Gō-like models and those of real proteins, but they do help to clarify key topological and energetic assumptions that are commonly used to justify such caricatures.     

Tryptophan-tryptophan energy migration as a tool to follow apoflavodoxin folding

Submolecular details of Azotobacter vinelandii apoflavodoxin (apoFD) (un)folding are revealed by time-resolved fluorescence anisotropy using wild-type protein and variants lacking one or two of apoFD's three tryptophans. ApoFD equilibrium (un)folding by guanidine hydrochloride follows a three-state model: native ↔ unfolded ↔ intermediate. In native protein, W128 is a sink for Förster resonance energy transfer (FRET). Consequently, unidirectional FRET with a 50-ps transfer correlation time occurs from W167 to W128. FRET from W74 to W167 is much slower (6.9 ns). In the intermediate, W128 and W167 have native-like geometry because the 50-ps transfer time is observed. However, non-native structure exists between W74 and W167 because instead of 6.9 ns the transfer correlation time is 2.0 ns. In unfolded apoFD this 2.0-ns transfer correlation time is also detected. This decrease in transfer correlation time is a result of W74 and W167 becoming solvent accessible and randomly oriented toward one another. Apparently W74 and W167 are near-natively separated in the folding intermediate and in unfolded apoFD. Both tryptophans may actually be slightly closer in space than in the native state, even though apoFD's radius increases substantially upon unfolding. In unfolded apoFD the 50-ps transfer time observed for native and intermediate folding states becomes 200 ps as W128 and W167 are marginally further separated than in the native state. Apparently, apoFD's unfolded state is not a featureless statistical coil but contains well-defined substructures. The approach presented is a powerful tool to study protein folding.     

Refolding simulations of an isolated fragment of barnase into a native-like β hairpin: Evidence for compactness and hydrogen bonding as concurrent stabilizing factors

Experimental evidence and theoretical models both suggest that protein folding is initiated within specific fragments intermittently adopting conformations close to that found in the protein native structure. These folding initiation sites encompassing short portions of the protein are ideally suited for study in isolation by computational methods aimed at peering into the very early events of folding. We have used Molecular Dynamics (MD) technique to investigate the behavior of an isolated protein fragment formed by residues 85 to 102 of barnase that folds into a β hairpin in the protein native structure. Three independent MD simulations of 1.3 to 1.8 ns starting from unfolded conformations of the peptide portrayed with an all-atom model in water were carried out at gradually decreasing temperature. A detailed analysis of the conformational preferences adopted by this peptide in the course of the simulations is presented. Two of the unfolded peptide conformations fold into a hairpin characterized by native and a larger bulk of nonnative interactions. Both refolding simulations substantiate the close relationship between interstrand compactness and hydrogen bonding network involving backbone atoms. Persistent compactness witnessed by side-chain interactions always occurs concomitantly with the formation of backbone hydrogen bonds. No highly populated conformations generated in a third simulation starting from the remotest unfolded conformer relative to the native structure are observed. However, nonnative long-range and medium-range contacts with the aromatic moiety of Trp94 are spotted, which are in fair agreement with a former nuclear magnetic resonance study of a denaturing solution of an isolated barnase fragment encompassing the β hairpin. All this lends reason to believe that the 85–102 barnase fragment is a strong initiation site for folding. Proteins 29:212–227, 1997. © 1997 Wiley-Liss, Inc.     

New structural insights into the refolding of ribonuclease T1 as seen by time-resolved Fourier-transform infrared spectroscopy

To get new structural insights into different phases of the renaturation of ribonuclease T1 (RNase T1), the refolding of the thermally unfolded protein was initiated by rapid temperature jumps and detected by time-resolved Fourier-transform infrared spectroscopy. The characteristic spectral changes monitoring the formation of secondary structure and tertiary contacts were followed on a time scale of 10(-3) to 10(3) seconds permitting the characterization of medium and slow folding reactions. Additionally, structural information on the folding events that occurred within the experimental dead time was indirectly accessed by comparative analysis of kinetic and steady-state refolding data. At slightly destabilizing refolding temperatures of 45 degrees C, which is close to the unfolding transition region, no specific secondary or tertiary structure is formed within 180 ms. After this delay all infrared markers bands diagnostic for individual structural elements indicate a strongly cooperative and relatively fast folding, which is not complicated by the accumulation of intermediates. At strongly native folding temperatures of 20 degrees C, a folding species of RNase T1 is detected within the dead time, which already possesses significant amounts of antiparallel beta-sheets, turn structures, and to some degree tertiary contacts. The early formed secondary structure is supposed to comprise the core region of the five-stranded beta-sheet. Despite these nativelike characteristics the subsequent refolding events are strongly heterogeneous and slow. The refolding under strongly native conditions is completed by an extremely slow formation or rearrangement of a locally restricted beta-sheet region accompanied by the further consolidation of turns and denser backbone packing. It is proposed that these late events comprise the final packing of strand 1 (residues 40-42) of the five-stranded beta-sheet against the rest of this beta-sheet system within an otherwise nativelike environment. This conclusion was supported by the comparison of refolding of RNase T1 and its variant W59Y RNase T1 that enabled the assignment of these very late events to the trans-->cis isomerization reaction of the prolyl peptide bond preceding Pro-39.     

Non-native interactions play an effective role in protein folding dynamics

Systematic Monte Carlo simulations of simple lattice models show that the final stage of protein folding is an ordered process where native contacts get locked (i.e., the residues come into contact and remain in contact for the duration of the folding process) in a well‐defined order. The detailed study of the folding dynamics of protein‐like sequences designed as to exhibit different contact energy distributions, as well as different degrees of sequence optimization (i.e., participation of non‐native interactions in the folding process), reveals significant differences in the corresponding locking scenarios—the collection of native contacts and their average locking times, which are largely ascribable to the dynamics of non‐native contacts. Furthermore, strong evidence for a positive role played by non‐native contacts at an early folding stage was also found. Interestingly, for topologically simple target structures, a positive interplay between native and non‐native contacts is observed also toward the end of the folding process, suggesting that non‐native contacts may indeed affect the overall folding process. For target models exhibiting clear two‐state kinetics, the relation between the nucleation mechanism of folding and the locking scenario is investigated. Our results suggest that the stabilization of the folding transition state can be achieved through the establishment of a very small network of native contacts that are the first to lock during the folding process.     

TOUCHSTONEX: protein structure prediction with sparse NMR data

TOUCHSTONEX, a new method for folding proteins that uses a small number of long-range contact restraints derived from NMR experimental NOE (nuclear Overhauser enhancement) data, is described. The method employs a new lattice-based, reduced model of proteins that explicitly represents C(alpha), C(beta), and the sidechain centers of mass. The force field consists of knowledge-based terms to produce protein-like behavior, including various short-range interactions, hydrogen bonding, and one-body, pairwise, and multibody long-range interactions. Contact restraints were incorporated into the force field as an NOE-specific pairwise potential. We evaluated the algorithm using a set of 125 proteins of various secondary structure types and lengths up to 174 residues. Using N/8 simulated, long-range sidechain contact restraints, where N is the number of residues, 108 proteins were folded to a C(alpha)-root-mean-square deviation (RMSD) from native below 6.5 A. The average RMSD of the lowest RMSD structures for all 125 proteins (folded and unfolded) was 4.4 A. The algorithm was also applied to limited experimental NOE data generated for three proteins. Using very few experimental sidechain contact restraints, and a small number of sidechain-main chain and main chain-main chain contact restraints, we folded all three proteins to low-to-medium resolution structures. The algorithm can be applied to the NMR structure determination process or other experimental methods that can provide tertiary restraint information, especially in the early stage of structure determination, when only limited data are available.     

Non-native interactions,effective contact order,and protein folding: a mutational investigation with the energetically frustrated hydrophobic model

By Monte Carlo simulations, we explored the effect of single mutations on the thermodynamics and kinetics of the folding of a two-dimensional, energetically frustrated, hydrophobic protein model. Phi-Value analysis, corroborated by simulations beginning from given sets of judiciously chosen initial contacts, suggests that the transition state of the model consists of a limited region of the native structure, that is, a folding nucleus. It seems that the most important contacts in the transition state (large and positive Phi) are not the ones with the highest contact order, because in this case the entropic cost of their formation would be too high, but exactly the ones that decrease the entropic cost of difficult contacts, reducing their effective contact order. Mutations of internal monomers involved in high-order contacts were actually the ones resulting in the fastest kinetics (and Phi < 0), indicating they tend to make low order, non-native contacts of low entropic cost that stabilize the unfolded state with respect to the transition state. Folding acceleration by other non-native interactions was also observed and a simple general mechanism is proposed according to which non-native contacts can act indirectly over the folding nucleus, "chelating" out potentially harmful contacts. The polymer graph of our model, which facilitates the visualization of effective contact orders, successfully suggests the relative kinetic importance of different contacts and is reasonably consistent with analogous graphs for the well characterized family of SH3 domains.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

Low folding cooperativity of HP35 revealed by single-molecule force spectroscopy and molecular dynamics simulation

Some small proteins, such as HP35, fold at submicrosecond timescale with low folding cooperativity. Although these proteins have been extensively investigated, still relatively little is known about their folding mechanism. Here, using single-molecule force spectroscopy and steered molecule dynamics simulation, we study the unfolding of HP35 under external force. Our results show that HP35 unfolds at extremely low forces without a well-defined unfolding transition state. Subsequently, we probe the structure of unfolded HP35 using the persistence length obtained in the force spectroscopy. We found that the persistence length of unfolded HP35 is around 0.72 nm, >40% longer than typical unstructured proteins, suggesting that there are a significant amount of residual secondary structures in the unfolded HP35. Molecular dynamics simulation further confirmed this finding and revealed that many native contacts are preserved in HP35, even its two ends have been extended up to 8 nm. Our results therefore suggest that retaining a significant amount of secondary structures in the unfolded state of HP35 may be an efficient way to reduce the entropic cost for the formation of tertiary structure and increase the folding speed, although the folding cooperativity is compromised. Moreover, we anticipate that the methods we used in this work can be extended to the study of other proteins with complex folding behaviors and even intrinsically disordered ones.     

Kinetics of intramolecular contact formation in a denatured protein

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule F?rster resonance energy transfer (FRET) experiments.     

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high‐energy conformation susceptible to proteolysis (cleavable form) using native‐state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m‐value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F‐G and Met‐20 loops on one face of the central β‐sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near‐native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F‐G and Met‐20 loops, which is coupled with compaction of the rest of the protein.     

Dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding

The rate of formation of intramolecular interactions in unfolded proteins determines how fast conformational space can be explored during folding. Characterization of the dynamics of unfolded proteins is therefore essential for the understanding of the earliest steps in protein folding. We used triplet-triplet energy transfer to measure formation of intrachain contacts in different unfolded polypeptide chains. The time constants (1/k) for contact formation over short distances are almost independent of chain length, with a maximum value of about 5 ns for flexible glycine-rich chains and of 12 ns for stiffer chains. The rates of contact formation over longer distances decrease with increasing chain length, indicating different rate-limiting steps for motions over short and long chain segments. The effect of the amino acid sequence on local chain dynamics was probed by using a series of host-guest peptides. Formation of local contacts is only sixfold slower around the stiffest amino acid (proline) compared to the most flexible amino acid (glycine). Good solvents for polypeptide chains like EtOH, GdmCl and urea were found to slow intrachain diffusion and to decrease chain stiffness. These data allow us to determine the time constants for formation of the earliest intrachain contacts during protein folding.     

Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins

Current theoretical views of the folding process of small proteins (< approximately 100 amino acids) postulate that the landscape of potential mean force (PMF) for the formation of the native state has a funnel shape and that the free energy barrier to folding arises from the chain configurational entropy only. However, recent theoretical studies on the formation of hydrophobic clusters with explicit water suggest that a barrier should exist on the PMF of folding, consistent with the fact that protein folding generally involves a large positive activation enthalpy at room temperature. In addition, high-resolution structural studies of the hidden partially unfolded intermediates have revealed the existence of non-native interactions, suggesting that the correction of the non-native interactions during folding should also lead to barriers on PMF. To explore the effect of a PMF barrier on the folding behavior of proteins, we modified Zwanzig's model for protein folding with an uphill landscape of PMF for the formation of transition states. We found that the modified model for short peptide segments can satisfy the thermodynamic and kinetic criteria for an apparently two-state folding. Since the Levinthal paradox can be solved by a stepwise folding of short peptide segments, a landscape of PMF with a locally uphill search for the transition state and cooperative stabilization of folding intermediates/native state is able to explain the available experimental results for small proteins. We speculate that the existence of cooperative hidden folding intermediates in small proteins could be the consequence of the highly specific structures of the native state, which are selected by evolution to perform specific functions and fold in a biologically meaningful time scale.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.