Lack of definable nucleation sites in the rate-limiting transition state of barnase under native conditions.

It has been shown that the burst-phase (submillisecond) intermediate of barnase, if it exists, can be only marginally more stable than the fully unfolded state at pH 6.3 and 25 degrees C. In the study reported here, no stable burst-phase intermediate could be detected, even in the presence of stabilizing salt (0.4 M Na(2)SO(4)). These results suggest that a burst-phase intermediate with even marginal stability does not exist. The absence of such an intermediate in turn suggests the need for re-examination of the rate-limiting transition state (RLTS) under native conditions, which was previously characterized by using a three-state model with a stable intermediate and protein engineering. Surprisingly, mutations throughout the structure of barnase do not significantly affect the folding rate, suggesting a lack of specific favorable interactions among the side-chains in the RLTS. This RLTS is clearly different from that previously characterized under denaturing conditions, indicating that changes take place in the RLTS under native and denaturing conditions. The occurrence of such changes is further supported by the observation that the unfolding rate constants of barnase and its mutants were divergent or convergent as a function of denaturant concentrations. Consistent with changes in the RLTS, a re-analysis of data from native-state hydrogen exchange studies has shown that the logarithm of the unfolding rate constant inflects down under low concentrations of denaturant. Here, we discuss in detail the question of whether changes in the RLTS involve a kinetically silent intermediate that occurs after the initial RLTS.     

The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate

Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rd-apocytochrome b(562) and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the N-terminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many non-native side-chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.     

The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.     

The kinetic pathway of folding of barnase

To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.     

An on-pathway hidden intermediate and the early rate-limiting transition state of Rd-apocytochrome b562 characterized by protein engineering

The folding pathway of Rd-apocytochrome b562, a four-helix bundle protein, was characterized using Trp and Ala/Gly pair mutations. We found that the Trp mutants (F65W) of both the fully folded Rd-apocytochrome b562 and a partially unfolded intermediate with the N-terminal helix (helix I) unfolded, fold with identical folding rates, providing direct evidence for the conclusion that the rate-limiting transition state folds before the partially unfolded intermediate; and that this hidden intermediate is an on-pathway intermediate. We further characterized the helical structures formed in the rate-limiting transition state by measuring the folding/unfolding rates for Ala/Gly pair mutations at solvent-exposed positions. Little change in folding rates occurred for the Ala/Gly pair mutations at positions in helix I and the C-terminal regions of helix II and IV. In contrast, a significant difference in folding rates was observed for the Ala/Gly pair mutations in helix III and the N-terminal regions of helix II and IV, suggesting that helix III and the N-terminal regions of helix II and IV are formed in the rate-limiting transition state. These results complement those obtained from earlier studies and help to define the folding pathway of Rd-apocytochrome b562 in more detail.     

The denatured state under native conditions: a non-native-like collapsed state of N-PGK

The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character. At denaturant concentrations immediately above the mid-point of unfolding, size-exclusion chromatography shows N-PGK to have a compact, denatured form, suggesting that it forms a helical molten globule. Within this globule, the helices extend into some regions that become beta strands in the native state. This predisposition of the denatured state to extensive non-native-like conformation, illustrates that, rather than directing folding, conformational pre-organization in the denatured state can compete with the normal folding direction. The corresponding reduction in control of the direction of folding as proteins become larger, could thus constitute a restriction on the size of protein domains.     

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.     

The folding of an enzyme. VI. The folding pathway of barnase: comparison with theoretical models.

The sequence of events in the refolding pathway of barnase has been analysed to search for general principles in protein folding. There appears to be a correlation between burying hydrophobic surface area and early folding events. All the regions that fold early interact extensively with the beta-sheet. These interactions involve predominantly hydrophobic interactions and the burial of very extensive hydrophobic areas in which multiple, close, hydrophobic-hydrophobic contacts are established around a central group of aliphatic residues. There is no burial of hydrophilic residues in these regions; those that are partly screened from the solvent make hydrogen bonds. All the regions or interactions that are made late in the folding pathway do not make extensive contacts with the beta-sheet. Their buried hydrophobic regions lack a central hydrophobic residue or residues around which other hydrophobic residues pack. Further, in some of these regions there is an extensive burial of hydrophilic residues. The results are consistent with one of the earlier events in protein folding being the local formation of native-like secondary structure elements driven by local hydrophobic surface burial. A possible candidate for an initiation site is a beta-hairpin between beta-strands 3 and 4 that is conserved in the microbial ribonuclease family. A comparison of structures in this family shows that those regions that can be superimposed, or have sequence homology, correspond to elements of structure that are formed and interact with each other early in the folding pathway, suggesting that some of these residues could be involved in directing the folding process. The data on barnase combined with results from other laboratories suggest the following tentative conclusions for the refolding of small monomeric proteins. (1) The refolding pathway is, at least in part, sequential and of compulsory order. (2) Secondary structure formation is driven by local hydrophobic surface burial and precedes the formation of most tertiary interactions. These elements are then stabilized and sometimes elongated by tertiary interactions. It is plausible that there are stop signals encoded in the linear sequence that prevent the elongation of isolated secondary structure elements in solution to a larger extent than is found in the folded protein. (3) Many tertiary interactions are not very constrained in the intermediate but become more and more defined as the hydrophobic cores consolidate, loop structures form and the configuration of surface residues takes place. The interactions between different elements of secondary structure are the last ones to be consolidated while the interactions within the secondary structure elements are consolidated earlier.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.     

Detection of a hidden folding intermediate in the focal adhesion target domain: Implications for its function and folding

The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

The denatured state of HIV-1 protease under native conditions

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process.     

The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure.

The structure of the first significant transition state on the unfolding pathway of barnase has been analysed in detail by protein engineering methods. Over 50 mutations placed strategically over the whole protein have been used as probes to report on the local structure in the transition state. Several different probes for many regions of the protein give consistent results as do multiple probes at the same site. The overall consistency of phi values indicates that the mutations have not produced changes in the protein that significantly alter the transition state for unfolding. A fine-structure analysis of interactions has also been conducted by removing different parts of the same side-chains. Many of the results of simple mutations fall nicely into the two clear-cut cases of phi = 1 or 0, indicating that the local noncovalent bonds are either fully broken or fully made in the transition state. Much of the structure of barnase in the transition state for unfolding is very similar to that in the folded protein. Both major alpha-helices fray at the N terminus. The last two turns in helix1 are certainly intact, as is the C terminus of helix2. The general picture of the beta-sheet is that the three central beta-strands are completely intact while the two edge beta-strands are mainly present but certainly weakened. The first five residues of the protein unwind but the C terminus remains folded. Three of the five loops are unfolded. The edges of the main hydrophobic core (core1) are significantly weakened, however, and their breaking appears partly rate determining. The centre of the small hydrophobic core3 remains intact. Core2 is completely disrupted. The first events in unfolding are thus: the unfolding of several loops, the unwinding of the helices from the N termini, and the weakening and disruption of the hydrophobic cores. The values of phi are found to be substantially the same under conditions that favour folding as under conditions that are highly denaturing, and so the structure of the unfolding transition state is substantially the same in water as in the presence of denaturant. The structure of the final kinetically significant transition state for refolding is identical to that for unfolding. The final events in refolding are, accordingly, the consolidation of the hydrophobic cores, the closing of many loops and the capping of the N termini of the helices.     

Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway

Intermediates along a protein's folding pathway can play an important role in its biology. Previous kinetics studies have revealed an early folding intermediate for T4 lysozyme, a small, well-characterized protein composed of an N-terminal and a C-terminal subdomain. Pulse-labeling hydrogen exchange studies suggest that residues from both subdomains contribute to the structure of this intermediate. On the other hand, equilibrium native state hydrogen experiments have revealed a high-energy, partially unfolded form of the protein that has an unstructured N-terminal subdomain and a structured C-terminal subdomain. To resolve this discrepancy between kinetics and equilibrium data, we performed detailed kinetics analyses of the folding and unfolding pathways of T4 lysozyme, as well as several point mutants and large-scale variants. The data support the argument for the presence of two distinct intermediates, one present on each side of the rate-limiting transition state barrier. The effects of circular permutation and site-specific mutations in the wild-type and circular permutant background, as well as a fragment containing just the C-terminal subdomain, support a model for the unfolding intermediate with an unfolded N-terminal and a folded C-terminal subdomain. Our results suggest that the partially unfolded form identified by native state hydrogen exchange resides on the folded side of the rate-limiting transition state and is, therefore, under most conditions, a "hidden" intermediate.     

The elusive intermediate on the folding pathway of the prion protein

A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrP(C)) to an altered disease-associated form, generally denoted as scrapie isoform (PrP(Sc)). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrP(C) may be recruited to form PrP(Sc). In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed.     

Three-state kinetic folding mechanism of the H2A/H2B histone heterodimer: the N-terminal tails affect the transition state between a dimeric intermediate and the native dimer

The H2A/H2B heterodimer is a component of the nucleosome core particle, the fundamental repeating unit of chromatin in all eukaryotic cells. The kinetic folding mechanism for the H2A/H2B dimer has been determined from unfolding and refolding kinetics as a function of urea using stopped-flow, circular dichroism and fluorescence methods. The kinetic data are consistent with a three-state mechanism: two unfolded monomers associate to form a dimeric intermediate in the dead-time of the SF instrument (approximately 5 ms); this intermediate is then converted to the native dimer by a slower, first-order reaction. Analysis of the burst-phase amplitudes as a function of denaturant indicates that the dimeric kinetic intermediate possesses approximately 50% of the secondary structure and approximately 60% of the surface area burial of the native dimer. The stability of the dimeric intermediate is approximately 30% of that of the native dimer at the monomer concentrations employed in the SF experiments. Folding-to-unfolding double-jump experiments were performed to monitor the formation of the native dimer as a function of folding delay times. The double-jump data demonstrate that the dimeric intermediate is on-pathway and obligatory. Formation of a transient dimeric burst-phase intermediate has been observed in the kinetic mechanism of other intertwined, segment-swapped, alpha-helical, DNA-binding dimers, such as the H3-H4 histone dimer, Escherichia coli factor for inversion stimulation and E.coli Trp repressor. The common feature of a dimeric intermediate in these folding mechanisms suggests that this intermediate may accelerate protein folding, when compared to the folding of archael histones, which do not populate a transient dimeric species and fold more slowly.     

The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.     

Group II intron folding under near-physiological conditions: collapsing to the near-native state

The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 degrees C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions. We observe that compaction of the RNA proceeds slowly to completion, even at low magnesium concentration (3 mM). Kinetic analysis shows that this compact species is a "near-native" intermediate state that is readily chased into the native state by the addition of high salt. Structural probing reveals that the near-native state represents a compact domain 1 scaffold that is not yet docked with the catalytic domains (D3 and D5). Interestingly, native ribozyme reverts to the near-native state upon reduction in magnesium concentration. Therefore, while the intron can sustain the intermediate state under physiological conditions, the native structure is not maintained and is likely to require stabilization by protein cofactors in vivo.