Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

On the origin of the cooperativity of protein folding: Implications from model simulations

There is considerable experimental evidence that the cooperativity of protein folding resides in the transition from the molten globule to the native state. The objective of this study is to examine whether simplified models can reproduce this cooperativity and if so, to identify its origin. In particular, the thermodynamics of the conformational transition of a previously designed sequence (A. Kolinski, W. Galazka, and J. Skolnick, J. Chem. Phys. 103: 10286–10297, 1995), which adopts a very stable Greek-key β-barrel fold has been investigated using the entropy Monte Carlo sampling (ESMC) technique of Hao and Scheraga (M.-H. Hao and H.A. Scheraga, J. Phys. Chem. 98: 9882–9883, 1994). Here, in addition to the original potential, which includes one body and pair interactions between side chains, the force field has been supplemented by two types of multi-body potentials describing side chain interactions. These potentials facilitate the proteinlike pattern of side chain packing and consequently increase the cooperativity of the folding process. Those models that include an explicit cooperative side chain packing term exhibit a well-defined all-or-none transition from a denatured, random coil state to a high-density, well-defined, nativelike low-energy state. By contrast, models lacking such a term exhibit a conformational transition that is essentially continuous. Finally, an examination of the conformations at the free-energy barrier between the native and denatured states reveals that they contain a substantial amount of native-state secondary structure, about 50% of the native contacts, and have an average root mean square radius of gyration that is about 15% larger than native. © 1996 Wiley-Liss, Inc.     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein G.

The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time (< or = 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 degrees C.     

Water as a conformational editor in protein folding

As molecules approach one another in aqueous solution, desolvation free energy barriers to association are encountered. Experiments suggest these (de)solvation effects contribute to the free energy barriers separating the folded and unfolded states of protein molecules. To explore their influence on the energy landscapes of protein folding reactions, we have incorporated desolvation barriers into a semi-realistic, off-lattice protein model that uses a simplified physico-chemical force-field determined solely by the sequence of amino acids. Monte Carlo sampling techniques were used to study the effects on the thermodynamics and kinetics of folding of a number of systems, diverse in structure and sequence. In each case, desolvation barriers increase the stability of the native conformation and the cooperativity of the major folding/unfolding transition. The folding times of these systems are reduced significantly upon inclusion of desolvation barriers, demonstrating that the particulate nature of the solvent engenders a more defined route to the native fold.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions

The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.     

Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions

The N-terminal beta-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 is also significantly populated in the transition state (phi(F)-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Ubeta4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.     

Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding

Do stable intermediates form very early in the protein folding process? New results and a quantity of literature that bear on this issue are examined here. Results available provide little support for early intermediate accumulation before an initial search-dependent nucleation barrier.     

Destabilization of the Escherichia coli RNase H kinetic intermediate: switching between a two-state and three-state folding mechanism

Escherichia coli RNase H folds through a partially folded kinetic intermediate that mirrors a rarely populated, partially unfolded form detectable by native-state hydrogen exchange under equilibrium conditions. Residue 53 is at the interface of two helices known to be structured in this intermediate. Kinetic refolding studies on mutant proteins varying in size and hydrophobicity at residue 53 support a contribution of hydrophobicity to the stabilities of the kinetic intermediate and the transition state. Packing interactions also play a significant role in the stability of these two states, though they play a much larger role in the native-state stability. One dramatic mutation, I53D, results in the conversion from a three-state to a two-state folding mechanism, which is explained most easily through a simple destabilization of the kinetic intermediate such that it is no longer stable with respect to the unfolded state. These results demonstrate that interactions that stabilize an intermediate can accelerate folding if these same interactions are present in the transition state. Our results are consistent with a hierarchical model of folding, where the intermediate consists of native-like interactions, is on-pathway, and is productive for folding.     

Rapid collapse precedes the fast two-state folding of the cold shock protein

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by F?rster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.