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The BCL-2 family of proteins plays a central role in regulating cell survival and apoptosis. Disordered BH3-only proteins bind promiscuously to a number of different BCL-2 proteins, with binding affinities that vary by orders of magnitude. Here we investigate the basis for these differences in affinity. We show that eight different disordered BH3 proteins all bind to their BCL-2 partner (MCL-1) very rapidly, and that the differences in sequences result in different dissociation rates. Similarly, mutation of the binding surface of MCL-1 generally affects association kinetics in the same way for all BH3 peptides but has significantly different effects on the dissociation rates. Importantly, we infer that the evolution of homologous, competing interacting partners has resulted in complexes with significantly different lifetimes.

Fibres were electrospun from blends of an epoxidized natural rubber (ENR) with a minor amount of a crystalline grade of polylactic acid (PLA), using a graft copolymer compatibilizer (ENR-g-JM) produced by reaction processing of a mixture of PLA and monoamine terminated polypropylene glycol (Jeffamine M600). The incorporation of PLA into the elastomer spinning solution in the form of a blend was necessary to obtain the required solution properties and to establish the appropriate operational conditions for the successful electrospinning of fibres. The addition of a small quantity of compatibilizer to the ENR/PLA blend reduced the severity of surface roughness of the fibres. Moreover, the use of monoamine terminated polypropylene glycol alone, as a plasticizer, was also found to exert a control on the development of surface texture during electrospinning. The rate of solvent induced crystallization in the swollen fibres jet was identified as the factor determining the surface topography.

Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape., Graphical Abstract, Highlights • Homologous spectrin domains have very different folding kinetics and mechanisms. • R15 has been engineered to fold and unfold slowly similar to R16 and R17. • The folding pathway is entirely unchanged. • R15 is a rare example of a protein with an inflexible transition state.

Many intrinsically disordered proteins (IDPs) are significantly unstructured under physiological conditions. A number of these IDPs have been shown to undergo coupled folding and binding reactions whereby they can gain structure upon association with an appropriate partner protein. In general, these systems display weaker binding affinities than do systems with association between completely structured domains, with micromolar Kd values appearing typical. One such system is the association between α- and β-spectrin, where two partially structured, incomplete domains associate to form a fully structured, three-helix bundle, the spectrin tetramerization domain. Here, we use this model system to demonstrate a method for fitting association and dissociation kinetic traces where, using typical biophysical concentrations, the association reactions are expected to be highly reversible. We elucidate the unusually slow, two-state kinetics of spectrin assembly in solution. The advantages of studying kinetics in this regime include the potential for gaining equilibrium constants as well as rate constants, and for performing experiments with low protein concentrations. We suggest that this approach would be particularly appropriate for high-throughput mutational analysis of two-state reversible binding processes.

The studies of the folding of structurally related proteins have proved to be a very important tool for investigating protein folding. Here we review some of the insights that have been gained from such studies. Our highlighted studies show just how such an investigation should be designed and emphasise the importance of the synergy between experiment and theory. We also stress the importance of choosing the right system carefully, exploiting the excellent structural and sequence databases at our disposal.

Mutations in polycystin-1 (PC1) can cause autosomal dominant polycystic kidney disease, which is a leading cause of renal failure. The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary cilia, neighboring cells, and extracellular matrix. PC1 is a large membrane protein that has a long N-terminal extracellular region (about 3000 amino acids) with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains, which are targeted by many naturally occurring missense mutations. Nothing is known about the effects of these mutations on the biophysical properties of PKD domains. Here we investigate the effects of several naturally occurring mutations on the mechanical stability of the first PKD domain of human PC1 (HuPKDd1). We found that several missense mutations alter the mechanical unfolding pathways of HuPKDd1, resulting in distinct mechanical phenotypes. Moreover, we found that these mutations also alter the thermodynamic stability of a structurally homologous archaeal PKD domain. Based on these findings, we hypothesize that missense mutations may cause autosomal dominant polycystic kidney disease by altering the stability of the PC1 ectodomain, thereby perturbing its ability to sense mechanical signals.

Fracture in rubber, like in all other materials, is initiated at imperfections inadvertently present or introduced in the body of the material or on the surface during processing. The point of initiation of fracture in the rubber is usually at flaws which are macroscopic or microscopic imperfections or inhomogeneities in the rubber. In order to investigate the effect of these flaws on the properties of a natural rubber compound, cuts to mimic weld lines were introduced before and near scorch at various cure temperatures, and the time for a complete healing of these cuts determined. The tensile properties were not significantly affected by the introduction of weld lines before scorch provided sufficient time was allowed for healing. Healing time reduced with increase in cure temperature. The intrinsic flaw sizes in a material inherently affect the material tensile properties and depend significantly on the size of the flaw. Pinhole flaws of varying sizes introduced after cure had a significant effect on both tensile strength and fatigue life with the pinhole equivalent intrinsic flaw size estimated to be about 200 μm.

In order to elucidate the relative importance of secondary structure and topology in determining folding mechanism, we have carried out a phi-value analysis of the death domain (DD) from human FADD. FADD DD is a 100 amino acid domain consisting of six anti-parallel alpha helices arranged in a Greek key structure. We asked how does the folding of this domain compare with that of (a) other all-alpha-helical proteins and (b) other Greek key proteins? Is the folding pathway determined mainly by secondary structure or is topology the principal determinant? Our Φ-value analysis reveals a striking resemblance to the all-beta Greek key immunoglobulin-like domains. Both fold via diffuse transition states and, importantly, long-range interactions between the four central elements of secondary structure are established in the transition state. The elements of secondary structure that are less tightly associated with the central core are less well packed in both cases. Topology appears to be the dominant factor in determining the pathway of folding in all Greek key domains.

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th alpha-helical repeat from chicken brain alpha-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th alpha-helical repeat from chicken brain alpha-spectrin). An extensive Phi-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.

A number of β-sandwich immunoglobulin-like domains have been shown to fold using a set of structurally equivalent residues that form a folding nucleus deep within the core of the protein. Formation of this nucleus is sufficient to establish the complex Greek key topology of the native state. These nucleating residues are highly conserved within the immunoglobulin superfamily, but are less well conserved in the fibronectin type III (fnIII) superfamily, where the requirement is simply to have four interacting hydrophobic residues. However, there are rare examples where this nucleation pattern is absent. In this study, we have investigated the folding of a novel member of the fnIII superfamily whose nucleus appears to lack one of the four buried hydrophobic residues. We show that the folding mechanism is unaltered, but the folding nucleus has moved within the hydrophobic core.

Since user-friendly atomic force microscopes came onto the market a few years ago, scientists have explored the response of many proteins to applied force. This field has now matured beyond the phenomenological with exciting recent developments, particularly with regards to research into biological questions. For example, detailed mechanistic studies have suggested how mechanically active proteins perform their functions. Also, in vitro forced unfolding has been compared with in vivo protein import and degradation. Additionally, investigations have been carried out that probe the relationship between protein structure and response to applied force, an area that has benefited significantly from synergy between experiments and simulations. Finally, recent technological developments offer exciting new avenues for experimental studies.

Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain α-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-β immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.

It has proved impossible to purify some proteins implicated in disease in sufficient quantities to allow a biophysical characterization of the effect of pathogenic mutations. To overcome this problem we have analyzed 37 different disease-causing mutations located in the L1 and IL2Rgamma proteins in well characterized related model proteins in which mutations that are identical or equivalent to pathogenic mutations were introduced. We show that data from these models are consistent and that changes in stability observed can be correlated to severity of disease, to correct trafficking within the cell and to in vitro ligand binding studies. Interestingly, we find that any mutations that cause a loss of stability of more than 2 kcal/mol are severely debilitating, even though some model proteins with these mutations can be easily expressed and analyzed. Furthermore we show that the severity of mutation can be predicted by a DeltaDeltaG(evolution) scale, a measure of conservation. Our results demonstrate that model proteins can be used to analyze disease-causing mutations when wild-type proteins are not stable enough to carry mutations for biophysical analysis.

TI I27, a β-sandwich domain from the human muscle protein titin, has been shown to fold via two alternative pathways, which correspond to a change in the folding mechanism. Under physiological conditions, TI I27 folds by a classical nucleation-condensation mechanism (diffuse transition state), whereas at extreme conditions of temperature and denaturant it switches to having a polarized transition state. We have used experimental F-values as restraints in ensemble-averaged molecular dynamics simulations to determine the ensembles of structures representing the two transition states. The comparison of these ensembles indicates that when native interactions are substantially weakened, a protein may still be able to fold if it can access an alternative transition state characterized by a much larger entropic contribution. Analysis of the probability distribution of F-values derived from ensemble averaged simulations, enables us to identify residues that form contacts in some members of the ensemble but not in others illustrating that many interactions present in transition states are not strictly required for the successful completion of the folding process. © 2006 by the Biophysical Society.

We present an experimental and computational analysis of the folding pathway of the 17th domain of chicken brain alpha-spectrin, R17. Wild-type R17 folds in a two-state manner and the chevron plot (plot of the logarithm of the observed rate constant against concentration of urea) shows essentially linear folding and unfolding arms. A number of mutant proteins, however, show a change in slope of the unfolding arm at high concentration of denaturant, hinting at complexity in the folding landscape. Through a combination of mutational studies and high temperature molecular dynamics simulations we show that the folding of R17 can be described by a model with two sequential transition states separated by an intermediate species. The rate limiting transition state for folding in water has been characterized both through experimental Phi-value analysis and by simulation. In contrast, a detailed analysis of the transition state predicted to dominate under highly denaturing conditions is only possible by simulation.

Spectrin domains are three-helix bundles, commonly found in large tandem arrays. Equilibrium studies have shown that spectrin domains are significantly stabilized by their neighbors. In this work we show that domain:domain interactions can also have profound effects on their kinetic behavior. We have studied the folding of a tandem pair of spectrin domains (R1617) using a combination of single- and double-jump stopped flow experiments (monitoring folding by both circular dichroism and fluorescence). Mutant proteins were also used to investigate the complex folding kinetics. We find that, although the domains fold and unfold individually, there is a single rate-determining step for both folding and unfolding of the protein. This is consistent with the equilibrium observation of cooperative folding of the entire two-domain protein. The results may have important biological implications. Not only will the protein fold more efficiently during cotranslational folding, but the ability of the multidomain protein to withstand thermal unfolding in the cell will be dramatically increased. This study suggests that caution has to be exercised when extrapolating from single domains to larger proteins with a number of independently folding modules arranged in tandem. The multidomain protein spectrin is certainly more than “the sum of its parts”.

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.

Molecular dynamics simulations of the structurally homologous proteins TNfn3 and FNfn10 have been used to investigate the contributions to side-chain dynamics measured by NMR relaxation experiments. The results reproduce the variation in core side-chain dynamics observed by NMR and highlight the relevance of anharmonic motion and transitions between local minima for explaining NMR side-chain order parameters. A method is described for calculating converged order parameters by use of replica exchange molecular dynamics in conjunction with an implicit solvent model. These simulations allow the influence of various factors, such as the flexibility of side-chains and their free volume, on the mobility to be tested by perturbing the system. Deletion mutations are found to have the largest effect on the more densely packed FNfn10. Some counterintuitive effects are seen, such as an increase in order parameters close to deletion mutation sites, but these can be rationalized in terms of direct interactions with the modified side-chains. A statistical analysis of published order parameters supports the conclusions drawn from the simulations.

Studies on the folding of helical proteins have shown a wide range of different mechanisms and highlighted the importance of helical propensity as a factor in determining folding mechanism. Here, we contribute to this interesting field with the protein engineering phi-value analysis of the 16th domain of chicken brain alpha-spectrin, R16. The fortuitous curvature seen in the unfolding arm of the chevron plot allows us to investigate both early and late events in folding. R16 is the first two-state helical protein for which this has been possible.

In the "fold approach" proteins with a similar fold but different sequences are compared in order to investigate the relationship between native state structure and folding behaviour. Here we compare the properties of the transition states for folding of TI I27, the 27th immunoglobulin domain from human cardiac titin, and that of TNfn3, the third fibronectin type III domain from human tenascin. Experimental phi-values were used as restraints in molecular dynamics simulations to determine the structures that make up the transition state ensembles (TSEs) for folding of the two proteins. The restrained simulations that we present allow a detailed structural comparison of the two TSEs to be made. Further calculations show explicitly that for both proteins the formation of the interactions involving the residues in the folding nucleus is sufficient for the establishment of the topology of the Ig-like fold. We found that, although the folding nuclei of the two proteins are similar, the packing of the folding nucleus of TI I27 is much tighter than that of TNfn3, reflecting the higher experimental phi-values and beta(T) (Tanford Beta) of TI I27. These results suggest that the folding nucleus can be significantly deformed to accommodate extensive sequence variation while conserving the same folding mechanism.

Mechanical unfolding of proteins using atomic force microscopy is becoming a routine biophysical technique. Mechanistic investigations in this rapidly evolving field are beginning to resolve the factors that contribute to the behaviour of biological macromolecules under force. Here we describe the force-mode apparatus, the experimental set-up, tractable systems of study, and the analysis of the resultant force data. Finally we summarise some of the recent achievements and limitations of this technique.

Recent work has shown that a β-sandwich domain from the human muscle protein titin (TI I27) unfolds via more than one pathway, providing experimental evidence for a long-standing theoretical prediction in protein folding. Here we present a thermodynamic analysis of two transition states along different folding pathways for this protein. The unusual upwards curvature previously observed in the denaturant-dependent unfolding kinetics is increased at both high and low temperatures, indicating that the high denaturant pathway is becoming more accessible. The transition states in each pathway are structurally distinct and have very different heat capacities. Interestingly the nucleation–condensation pathway is dominant at all physiologically relevant temperatures, supporting the suggestion that pathways with diffuse rather than localised transition states have been selected for by evolution to prevent misfolding.

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.

The advent of a new class of force microscopes designed specifically to “pull” biomolecules has allowed non-specialists to use force microscopy as a tool to study single-molecule protein unfolding. This powerful new technique has the potential to explore regions of the protein energy landscape that are not accessible in conventional bulk studies. It has the added advantage of allowing direct comparison with single-molecule simulation experiments. However, as with any new technique, there is currently no well described consensus for carrying out these experiments. Adoption of standard schemes of data selection and analysis will facilitate comparison of data from different laboratories and on different proteins. In this review, some guidelines and principles, which have been adopted by our laboratories, are suggested. The issues associated with collecting sufficient high quality data and the analysis of those data are discussed. In single-molecule studies, there is an added complication since an element of judgement has to be applied in selecting data to analyse; we propose criteria to make this process more objective. The principal sources of error are identified and standardised methods of selecting and analysing the data are proposed. The errors associated with the kinetic parameters obtained from such experiments are evaluated. The information that can be obtained from dynamic force experiments is compared, both quantitatively and qualitatively to that derived from conventional protein folding studies.

The covalently linked domains constituting a multidomain protein typically share a similar fold and sequence, are generally stable in isolation, and are largely capable of independent folding. However, the elegant interplay of forces leading an amino acid sequence to its native state is more complex in proteins with multiple domains because of inter-domain interactions, which complicate the folding energy landscape.In previous experiments, employing immunoglobulin-like (Ig-like) domains of the human multidomain protein titin, we showed that tandem repeats of domains with high sequence identity can form a stable misfolded state upon refolding in physiological solution conditions. Conversely, tandem constructs of natural neighbouring domains, which display a low sequence identity, did not misfold. This supported the hypothesis that sequence identity between neighbouring domains in multidomain proteins is reduced as a result of evolutionary pressure to avoid misfolding.With a combination of single-molecule Forster resonance energy transfer, microfluidic mixing, stopped-flow kinetics and molecular dynamics simulations, we now demonstrate that Ig-like domains can transiently populate a surprisingly broad range of misfolded conformations on the sub-second timescale. Using tandem repeats of Ig-like domains, we can resolve both strand-swapped misfolds dominated by native-like interactions and, remarkably, a non-native-like, largely disordered type of misfolded state which so far was never observed experimentally, characterized by promiscuous interactions. Even more surprisingly, both types of misfolding are detected also for the naturally occurring tandem repeat, showing how finely the propensities of folding and misfolding have been balanced by co-evolution of adjacent domains to avoid stable misfolded states formation. On longer timescales, however, all or most of the protein molecules are able to reach the native state, demonstrating that the overall free energy surface is still sufficiently optimized for the protein to efficiently reach its correctly folded state.

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain-domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially "the sum of its parts". From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.

Anfinsen (1961) demonstrated that the information for a protein to fold is entirely contained within its sequence. More recently, the importance of unfolded, but functional, proteins has been recognised. These so-called intrinsically disordered proteins (IDPs) lack a three-dimensional structure, but may fold upon interacting with a partner protein. Where is the folding information for these coupled folding and binding reactions contained? Is the amino acid sequence of the IDP the sole determinant of the folding reaction? Or are they dictated by the partner protein? We examine these questions by comparing and contrasting multiple interactions between IDPs and their structured partners, utilising the Bcl-2 family as a model system.

Background: Do proteins that have the same structure fold by the same pathway even when they are unrelated in sequence? To address this question, we are comparing the folding of a number of different immunoglobulin-like proteins. Here, we present a detailed protein engineering φ value analysis of the folding pathway of TI I27, an immunoglobulin domain from human cardiac titin. Results: TI I27 folds rapidly via a kinetic intermediate that is destabilized by most mutations. The transition state for folding is remarkably native-like in terms of solvent accessibility. We use φ value analysis to map this transition state and show that it is highly structured; only a few residues close to the N-terminal region of the protein remain completely unfolded. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of obtaining additional structural information about the transition state. Conclusions: The residues that are involved in nucleating the folding of TI I27 are structurally equivalent to the residues that form the folding nucleus in an evolutionary unrelated fibronectin type III protein. These residues form part of the common structural core of Ig-like domains. The data support the hypothesis that interactions essential for defining the structure of these β sandwich proteins are also important in nucleation of folding.

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Brønsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is approximately 40 % of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different "layers" of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".

There has been a large body of work on the deposition of colloidal particles onto many different kinds and shapes of surfaces. Nearly all work in the past has involved flowing systems in which a well-defined hydrodynamic layer exists at the collector surface with certain advantages and disadvantages. We have chosen a still system in which transport of particles to the collector surface occurs solely by diffusion. Both particles and surfaces were negatively charged, so that deposition was driven by dispersion forces. A photomicroscopic technique has been used to determine absolute values of initial rates of deposition. Three monodisperse polystyrene colloids of various particle sizes and surface charge densities were synthesized. Their zeta potentials were determined by microelectrophoresis in Ba(NO 3 ) 2 solutions. Collector surfaces were glass and glass coated with polystyrene. Their zeta potentials were determined from their streaming potentials in various concentrations of electrolyte. Direct determination of diffusion coefficients indicate that the particles behave normally. Initial deposition rates exhibit strong dependence on ionic strength, indicating large electrical double layer effects. Like the work of others, rates of deposition fell off with time, perhaps due to the presence of `hot spots', i.e. highly localized areas of greater reactivity. The most regular behavior occurred in deposition on polystyrene-coated glass, presumably because roughness and highly reactive sites on the glass surface were largely eliminated. Absolute rates were several times theoretical for fastest deposition. It is speculated that the hydrophobic effect plays a significant role in accelerating particles towards the collector. Plots of log (initial rate constant) vs. log (ionic strength) were linear and generally in the order anticipated by Reerink–Overbeek theory.

The third fibronectin type III domain from human tenascin adopts a compact beta-sandwich fold. Its boundaries were originally selected to encode a 90-residue domain (TNfn31-90). We conclude that the dynamic properties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal (R1) and transverse (R2) 15N relaxation rates, and ¿1H-15N¿ NOE enhancements at pH 4.9 and 300 K are presented for TNfn31-90 and TNfn31-92, the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties over a broad range of timescales. However, a number of residues near the C terminus in TNfn31-90 exhibit elevated transverse relaxation rates and broadened signals in 1H-15N HSQC spectra. Explicit rates of chemical exchange for five residues in TNfn31-90 were determined by measuring transverse relaxation rates in a series of CPMG experiments with spin-echo refocusing delays increasing from 311 to 1436 micros. Calculated exchange rates average 1000(+/-311) s-1, with individual uncertainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn31-90 at pH 5. 64(+/-0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote chemical exchange in the shorter domain. Additionally, NOE and chemical shift data suggest hydrogen bond formation between the added residues and adjacent loops. The data affirm the importance of judiciously selecting domain boundaries prior to the characterization of molecular properties.

The structural and dynamic properties of an immunoglobulin superfamily domain (IgSF), Ig 18′, have been characterised by NMR at 285 K, in the presence of 4.2 M and 6.0 M urea, respectively. Analysis of chemical shift deviations, 3 J HNHα coupling constants, sequential NOE pattern, and 15 N relaxation data reveals that although the two urea-denatured states are highly disordered, some local turn-like residual structures do exist. Moreover, some distinct differences between the properties of the two denatured states are observed. In 4.2 M urea-denatured Ig 18′, regions 80–83 and 86–92 adopt turn-like conformations, furthermore, region 84–93 is involved in slow exchange processes that occur on a micro- to millisecond time-scale. In the 6.0 M urea-denatured state, these turn-like conformations are less occupied, and chemical exchange processes in region 84–93 are largely reduced. In contrast, region 32–36 has persistent turn-like structures in both urea-denatured states. Some correlation between the spectral density function at 0 frequency, J eff (0), for the urea-denatured states and the secondary structure elements of the folded state have been observed. Except for the terminal regions, residues corresponding to β-strands have higher J eff (0) values compared to residues corresponding to loops. The characterisation and comparison of the two urea-denatured states highlight residues that possess properties that may be crucial for the initiation of folding of this domain.

There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.

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.

IDPs are overrepresented in processes such as signalling and transcription, where proteins often interact with a range of partners. One much-studied key hub protein is the coactivator CBP/p300, whose folded KIX domain binds to a number of different intrinsically disordered transcription factors. The interaction of KIX with several of its ligands has been well studied by equilibrium methods, and structural information is available for many of the complexes. By careful control and consideration of experimental conditions such as temperature and ionic strength we have been able to perform kinetic studies that reveal the mechanism of the association reaction of KIX with cMyb; the fastest protein-protein interaction yet reported. Furthermore, through comparative studies with several binding partners we shed light on an important outstanding question in the IDP field: what is the advantage of disorder to a protein?View Large Image | View Hi-Res Image | Download PowerPoint Slide

Oligomerisation is essential for the function of some proteins. The PyrR family of proteins are involved in pyrimidine operon attenuation. They are regulated by the presence of certain nucleotides such as guanosine monophosphate (GMP), which stabilises the tetrameric state. Notably, some members of this family can adopt a tetrameric oligomerisation state regardless of the presence of the GMP. Perica et al. (2012) previously found that this family, among several others, have differences in sequence outside the oligomeric interfaces and these are sufficient to explain the changes to their subunit geometry and oligomerisation states. Here, we compared the differences in structural flexibility linked to the changes in oligomeric state caused by a) specific mutations and, b) by the presence of bound GMP. We calculated the coarse-grained normal modes of dimeric units of the PyrR dimers and tetramers using an Elastic Network Model implemented in the Molecular Modelling ToolKit. We conducted several analyses including comparing the normal modes of these proteins with each other using the Bhattacharyya Coefficient similarity measure, correlations matrices, and the conformational overlap analysis, by which the contribution of these modes to the transition from one state to another can be quantified. Firstly, we found that while the dynamics were very similar between all the structures, there was a noticeable difference between the dimeric and tetrameric units. We also show that both sets of proteins transition from tetrameric to dimeric states similarly, indicating some overlap between the effects of allostery and evolution on oligomerisation in PyrR.

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.

Backgound: Methods have been developed recently for probing local fluctuations of protein structure using H/ 2 H-exchange of amide protons at equilibrium. It has been suggested that equilibrium exchange methods can identify the order of events in folding pathways and detect folding cores. We have applied the procedure of measuring the effects of denaturant on the H/ 2 H-exchange of amide protons of barnase, the folding pathway of which is well established. Results The addition of relatively low concentrations of denaturant causes the mechanism of exchange of amide protons of barnase to change from EX2 to EX1 for the residues that require global unfolding for exchange to occur. This change of mechanism, which would have been missed by some of the standard tests, causes artefacts that could be easily misinterpreted. We also present the thermodynamic argument that measurements at equilibrium cannot give the order of events in folding. Conclusion Measurement of H/ 2 H-exchange of amide protons at equilibrium, when applied correctly, is an excellent method for analyzing the equilibrium distribution of unfolded and partly folded states. It cannot, in theory and in practice, be used for determining protein folding pathways by itself.

In this series of papers, we examine the effects of introducing disulfide bond on the properties, structure and thermodynamics of a small globular protein, barnase. Three mutants have been made, in each of which a single crosslink confers different properties. Two of the disulfide bonds, between residues 43 and 80 (43–80) and between residues 85 and 102 (85–102), stabilise the protein, relative to both wild-type and the corresponding (reduced) dithiol forms: 85–102 is more stable than predicted from the entropic destabilisation of the unfolded state; 43–80 is less stable than predicted. The third disulfide bond, between residues 70 and 92 (70–92) destabilises the protein relative to both wild-type and the corresponding dithiol form, implying significant disruption of the folded protein on formation of the disulfide bond. Crystal structures of three mutant proteins have been solved. All three proteins have essentially the same fold as wild-type, but with left-handed disulfide bonds, which have dihedral geometries that have not been observed in naturally occurring disulfides. In the very stable mutant 85–102, there is no significant difference between the mutant and wild-type structures: these data do not explain the large stability of this protein. The disulfide bond at 43–80 induces small structural rearrangements close to the site of the disulfide bond, associated with some local disorder: the crosslink appears to decrease the stability of the native form of the protein. The destabilizing disulfide bond at 70–92 induces considerable structural change, with displacement of a loop and consequent disruption of a stabilizing salt-bridge. Our studies do not support the view that the conformation of the disulfide bond is crucial in determining the stability of the mutant proteins.

Disorder in protein ranges from regions of the chain that are highly dynamic through to entirely unstructured regions or entire domains of larger proteins. This disorder can play a functional role, promoting cooperativity and imparting both structural integrity and allosteric coupling. I will describe some of our current studies of very different protein folding systems.