Weak cooperativity in the core causes a switch in folding mechanism between two proteins of the cks family.

The human protein ckshs1 (cks1) is a 79 residue alpha/beta protein with low thermodynamic and kinetic stability. Its folding mechanism was probed by mutation at sites throughout the structure. Many of the mutations caused changes in the slope of the unfolding arm of the chevron plot. The effects can be rationalised in terms of either transition-state movement or native-state "breathing", and in either case, the magnitude of the effect enables the sequence of events in the folding reaction to be determined. Those sites that fold early exhibit a small perturbation, whilst those sites that fold late exhibit a large perturbation. The results show that cks1 folds sequential pairs of beta-strands first; beta1/beta2 and beta3/beta4. Subsequently, these pairs pack against each other and onto the alpha-helical region to form the core. The folding process of cks1 contrasts with that of the homologue, suc1. The 113 residue suc1 has the same beta-sheet core structure but, additionally, two large insertions that confer much greater thermodynamic and kinetic stability. The more extensive network of tertiary interactions in suc1 provides sufficient enthalpic gain to overcome the entropic cost of forming the core and thus tips the balance in favour of non-local interactions: the non-local, central beta-strand pair, beta2/beta4, forms first and the periphery strands pack on later. Moreover, the greater cooperativity of the core of suc1 protects its folding from perturbation and consequently the slope of the unfolding arm of the chevron plot is much less sensitive to mutation.

Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1.

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.

Observation of signal transduction in three-dimensional domain swapping.

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues.

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer-dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

Sequence conservation provides the best prediction of the role of proline residues in p13suc1.

The unique nature of the proline side-chain imposes severe constraints on the polypeptide backbone, and thus it seems likely that it plays a special structural or functional role in the architecture of proteins. We have investigated the role of proline residues in suc1, a member of the cyclin-dependent kinase (cks) family of proteins, whose known function is to bind to and regulate the activity of the major mitotic cdk. The effect on stability of mutation to alanine of all but two of the eight proline residues is correlated with their conservation within the family. The remaining two proline residues are located in the hinge loop between two beta-strands that mediates a domain-swapping process involving exchange of a beta-strand between two monomers to form a dimer pair. Mutation of these proline residues to alanine stabilises the protein. cdk binding is unaffected by these mutations, but dimerisation is altered. We propose, therefore, that the double-proline motif is conserved for the purpose of domain swapping, which suggests that this phenomenon plays a role in the function of cks proteins. Thus, the conservation of the proline residues is a good indicator of their roles in suc1, either in the stabilisation of the native state or in performing functions that are as yet unknown. In addition, the strain resulting from two of the proline residues was relieved successfully by mutation of the preceeding residue to glycine, suggesting a general method for designing more stable proteins.

The folding pathway of the cell-cycle regulatory protein p13suc1: clues for the mechanism of domain swapping.

BACKGROUND: The 113-residue alpha+beta protein suc1 is a member of the cyclin-dependent kinase subunit (cks) family of proteins that are involved in regulation of the eukaryotic cell cycle. In vitro, suc1 undergoes domain swapping to form a dimer by the exchange of a C-terminal beta strand. We have analysed the folding pathway of suc1 in order to determine the atomic details of how strand-exchange occurs in vitro and thereby obtain clues as to the possible mechanism and functional role of dimerisation in vivo. RESULTS: The structures of the rate-determining transition state for the folding/unfolding of suc1 and of the intermediate that is populated during refolding were probed using phi values determined for 57 mutants with substitutions at 43 sites throughout the protein. The majority of phi values are fractional in the intermediate and transition state, indicating that interactions build up in a concerted manner during folding. In the transition state, phi values of greater than 0.5 are clustered around the inner strands beta2 and beta4 of the beta sheet. This part of the structure constitutes the nucleus for folding according to a nucleation-condensation mechanism. Molecular dynamics simulations of unfolding of suc1, performed independently in a blind manner, are in excellent agreement with experiment (proceeding paper). CONCLUSIONS: Strand beta4 is the exchanging strand in the dimer and yet it forms an integral part of the folding nucleus. This suggests that association is an early event in the folding reaction of the dimer. Therefore, interchange between the monomer and dimer must occur via an unfolded state, a process that may be facilitated in vivo by accessory proteins.

Stability and folding of the tumour suppressor protein p16.

The tumour suppressor p16 is a member of the INK4 family of inhibi tors of the cyclin D-dependent kinases, CDK4 and CDK6, that are involved in the key growth control pathway of the eukaryotic cell cycle. The 156 amino acid residue protein is composed of four ankyrin repeats (a helix-turn-helix motif) that stack linearly as two four-helix bundles resulting in a non-globular, elongated molecule. The thermodynamic and kinetic properties of the folding of p16 are unusual. The protein has a very low free energy of unfolding, Delta GH-2O/D-N, of 3.1 kcal mol-1 at 25 degreesC. The rate-determining transition state of folding/unfolding is very compact (89% as compact as the native state). The other unusual feature is the very rapid rate of unfolding in the absence of denaturant of 0.8 s-1 at 25 degreesC. Thus, p16 has both thermodynamic and kinetic instability. These features may be essential for the regulatory function of the INK4 proteins and of other ankyrin-repeat-containing proteins that mediate a wide range of protein-protein interactions. The mechanisms of inactivation of p16 by eight cancer-associated mutations were dissected using a systematic method designed to probe the integrity of the secondary structure and the global fold. The structure and folding of p16 appear to be highly vulnerable to single point mutations, probably as a result of the protein's low stability. This vulnerability provides one explanation for the striking frequency of p16 mutations in tumours and in immortalised cell lines.

Stability and folding of the cell cycle regulatory protein, p13(suc1).

p13(suc1) (suc1) is a member of the CDC28 kinase specific family of cell cycle regulatory proteins that bind to the cyclin-dependent kinase CDK2 and regulate its activity. suc1 has two distinct conformational and assembly states, a compact globular monomer and a beta strand-exchanged dimer. The dimerisation is an example of domain-swapping, and is mediated by a molecular hinge mechanism that is conserved across the entire CKS family. It has been proposed that the function of suc1 may be modulated by the dimerisation process with monomer-dimer switching occurring in response to a change in the cell environment. We have investigated the stability and folding of suc1 as a first step in determining the mechanism and functional role of the strand exchange. Suc1 unfolds reversibly at equilibrium in a two-state manner with a free energy of unfolding of 7.2 kcal mol-1. The kinetics of folding and unfolding are complex, and double-jump stopped-flow methods revealed that there are at least three parallel folding pathways arising from distinct unfolded and partly folded, intermediate states. The major population of unfolded species fold rapidly according to a three-state mechanism, D1->I1->N, with a rate constant for the formation of native species, N, from the intermediate, I1, of 65 s-1 in water. Two minor populations of unfolded molecules fold more slowly. Folding of one population is limited by proline isomerisation in a partly folded state, and some expansion of the protein is required for isomerisation to occur. The other population could be assigned to rate-limiting isomerisation of the peptidyl-proline bond of residue 90, which is located in the molecular hinge. A minor, fast phase was detected in the unfolding kinetics that corresponds to unfolding of a small population of a distinct native-like form. Heterogeneity was removed upon mutation of Pro90 to Ala. The unfolding kinetics of the strand-exchanged dimer were also investigated and showed that the dimer unfolds at the same rate as the monomer.

Synergy between simulation and experiment in describing the energy landscape of protein folding.

Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single alpha-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold (tau = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, Phi-values and molecular dynamics simulations, in particular, suggest that significant progress has been made toward describing complete folding pathways at atomic resolution by combining experiment and simulation.

Hydrogen exchange and protein folding.

Amide hydrogen-deuterium exchange is a sensitive probe of the structure, stability and dynamics of proteins. The significant increase in the number of small, model proteins that have been studied has allowed a better understanding of the structural fluctuations that lead to hydrogen exchange. Recent technical advances enable the methodology to be applied to the study of protein-protein interactions in much larger, more complex systems.

Complementation of peptide fragments of the single domain protein chymotrypsin inhibitor 2.

Chymotrypsin inhibitor 2 (CI2) folds kinetically as a single domain protein. It has been shown that elements of native secondary structure do not significantly form in fragments as the 64 residue protein is progressively increased in length from its N terminus, until at least 60 residues are present. Here, we analyse peptides of increasing length from the C terminus and find that native-like structure is not present even in the largest, fragment (7-64). We have examined sets of peptides of the form (1 - x) and ((x + 1)-64) to detect complementation. The only pair that readily complements and gives native-like structure is (1-40) and (41-64), where cleavage occurs in the protease-binding loop of CI2. But, all the pairs of peptides (1 - x) + (41-64) complement for x > 40, as do all pairs of (1-40) + (x-64), where x < 40. The resultant complexes appear to be equivalent to (1-40). (41-64) with the overlapping sequence being unstructured. Thus, the folding of CI2 is extremely co-operative, and interactions have to be made between subdomains (1-40) and (41-64). This is consistent with the mechanism proposed for the folding pathway of intact CI2 in which a diffuse nucleus is formed in the transition state between the alpha-helix in the N-terminal region of the protein and conserved hydrophobic contacts in the C-terminal region of the polypeptide. It is with these protein design features that CI2 can be an effective protease inhibitor.

Hydrogen exchange at equilibrium: a short cut for analysing protein-folding pathways?

Hydrogen exchange is an attractive method for observing small populations of partly unfolded states of proteins at equilibrium. It has been suggested that these represent folding intermediates so that hydrogen exchange can offer a short cut for studying protein-folding pathways. This cannot work in theory because it is not possible to tell whether they are intermediates or side reactions. Experimental studies of barnase and chymotrypsin inhibitor 2 show that there is no obvious relationship between hydrogen exchange at equilibrium and their folding pathways.

Hydrogen exchange in chymotrypsin inhibitor 2 probed by denaturants and temperature.

Hydrogen exchange of chymotrypsin inhibitor 2 has been measured in the presence of low concentrations of GdmCl and at different temperatures. The study of exchange at different temperatures allows us to obtain the activation enthalpies for the local exchange processes, and the change in enthalpy between the closed, exchange-incompetent, forms and the open, exchange-competent, forms. From the GdmCl dependence of exchange, an m-value, which is a measure of the new surface area exposed to solvent in the equilibrium between open and closed forms, can be determined for individual protons. This parameter therefore provides information about the structural nature of the opening reactions. In the absence of denaturant, exchange from native and native-like states dominates. As GdmCl concentration is increased, opening reactions that involve global unfolding are selectively promoted for the majority of amide protons. Three classes of protons emerge: for one set of protons, there is a linear and weak dependence on denaturant, indicating that the dominant opening reaction is the same throughout the range of GdmCl concentrations and involves local fluctuations with exposure of little new surface. For another set of protons, the most slowly exchanging residues, a linear, but much stronger, denaturant dependence is observed. For these protons, global unfolding dominates, and the m-values are similar to that obtained by equilibrium GdmCl denaturation measured by fluorescence under identical conditions. For the remaining protons, the GdmCl-dependence is weak at low GdmCl concentrations and increases at higher GdmCl concentrations. No segment of sub-global unfolding could be identified. Rather, all protons appear to merge together at high GdmCl concentrations to the global unfolding reaction.

Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis.

Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global unfolding or a mixture of the two pathways. For a large number of residues, the exchange rates were unaffected by mutations which destabilized the protein by up to 1.9 kcal mol(-1), indicating that exchange is occurring through local fluctuations of the native state. A small number of residues were found for which the mutations had the same effect on the rate constants for exchange as on the equilibrium constant for unfolding, indicating that these residues exchange by global unfolding. These are residues that have the slowest exchange rates in the wild-type protein. We see no correspondence between these residues and residues involved in the nucleation site for the folding reaction identified by protein engineering studies. Rather, the exchange behaviour of CI2 is determined by the native structure: the most protected amide protons are located in regions of hydrogen bonding, specifically the C terminus of the alpha-helix and the centre of the beta-sheet. A number of the most slowly exchanging residues are in the hydrophobic core of the protein.

Following co-operative formation of secondary and tertiary structure in a single protein module.

We have prepared a family of peptide fragments of the 64 amino acid protein chymotrypsin inhibitor (CI2), corresponding to progressive elongation from the N terminus, in order to elucidate the basis of conformational preferences in single-domain proteins and to obtain insights into their conformational pathway. Structural analysis of the fragment comprising the first 50 residues, CI2(1-50), indicates that it is mainly disordered, with patches of hydrophobic residues exposed to the solvent. Structural characterisation of the fragment CI2(1-63) which lacks only the C-terminal glycine, Gly64, shows native-like structure in all regions of the fragment. The study provides insights into the contribution of specific residues to the stability and co-operativity of the intact protein. We define a phiNMR value, derived from chemical shift analysis, which describes the build-up of structure at the level of individual residues (protons). All the macroscopic probes used to study the growth of structure in CI2 on elongation of the chain (circular dichroism, fluorescence and gel filtration) are in agreement with the residue-by-residue description by NMR. It is seen that secondary and tertiary structure build up in parallel in the fragments and show similar structures to those developed in the transition state for folding of the intact protein.

Strain in the folding nucleus of chymotrypsin inhibitor 2.

BACKGROUND: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single alpha helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles. RESULTS: Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state. CONCLUSIONS: Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.

Structure of the transition state for folding of a protein derived from experiment and simulation.

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the alpha-helix is weakened but partially intact and the beta-sheet is quite disrupted. As inferred from the phi f values and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a "folding core" comprising a patch on the alpha-helix and a portion of the beta-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived phi values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting phi F values and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.

Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.

Nature and consequences of GroEL-protein interactions.

The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL. The chaperonin retards the rate of refolding of wild type and 32 representative point mutants. The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on the mutation. It is seen qualitatively that truncation of large hydrophobic side chains to smaller side chains weakens binding. Analysis of the magnitude of the rates of retardation shows further that hydrophobic and positively charged side chains tend to interact favorably with GroEL whereas negatively charged side chains tend to repel. There is an inverse correlation between the strength of hydrophobic interactions and the rate constant for refolding of the GroEL-complexed protein: the better the binding, the slower the folding. This shows directly that hydrophobic (and other favorable) interactions between the chaperonin and substrate are weakened during the refolding process and implies that unfolding can be catalyzed by the gain of such interactions.

The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding.

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hydrophobic core, has its full native interaction energy in the transition state. The only region of structure which is well developed in the transition state is the alpha-helix (residues 12 to 24). But, the interactions within it are weakened, especially at the C-terminal region. The rest of the protein has varying degrees of weakly formed structure. Thus, secondary and tertiary interactions appear to form concurrently. These data, reinforced by studies on the structures of peptide fragments, fit a "nucleation-condensation" model in which the overall structure condenses around an element of structure, the nucleus, that itself consolidates during the condensation. The high energy transition state is composed of the whole of the molecule making a variety of weak interactions, the nucleus being those residues that make the strongest interactions. The nucleus here is part of the alpha-helix and some distant residues in the sequence with which it makes contacts. The remainder of the protein has to be sufficiently ordered that it provides the necessary interactions to stabilize the nucleus. The nucleus is only weakly formed in the denatured state but develops in the transition state. The onrush of stability as the nucleus consolidates its local and long range interactions is so rapid that it is not yet fully formed in the transition state. The formation of the nucleus is thus coupled with the condensation. These results are consistent with a recent simulation of the folding of a computer model protein on a lattice which is found to proceed by a nucleation-growth mechanism. We suggest that the mechanism of folding of CI2 may be a common theme in protein folding whereby fundamental folding units of larger proteins, which are modelled by the folding of CI2, form by nucleation-condensation events and coalesce, perhaps in a hierarchical manner.