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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.

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.

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.

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.

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”.

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.

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.

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.

SasG has a long repeat region made up of identical repeating E and G5 domains. Although the domains themselves are relatively unstable (indeed E domains on their own are unfolded), the cooperative folding of the domains results in formation of molecules that are long and remarkably mechanically resistant. We have used small angle X-ray scattering and mechanical unfolding methods, combined with simulations, to show that SasG constructs of physiological length are indeed monomeric, highly extended and mechanically strong. Obligate folding cooperativity of the intrinsically disordered E domain couples spatially separate G5 domains both thermodynamically and structurally, creating a superstructure that supersedes the domain architecture. Our findings provide a simple solution for the efficient assembly of mechano-resistant elongated structures of tunable length from a single polypeptide chain and have significant potential for the development of novel biomaterials.

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.

Although there have been many thermodynamic studies of the association of intrinsically disordered proteins (IDPs) with their binding partners, there have been relatively few kinetic studies.We have analysed the association of two different IDP systems, which form helical structure upon binding. These systems have markedly different behaviour. One associates very slowly and weakly, and the other very rapidly and strongly. The kinetic data therefore need to be analysed in a different manner to each other, and to the folding of single domain proteins. We discuss why standard approaches developed for folded protein-protein interactions may not be appropriate for reactions where one or both proteins are disordered. In addition, we show how protein engineering can be used to provide insights into the pathways/mechanisms of folding.Finally we have analysed the kinetics of the folding of an intrinsically disordered region (IDR) within a protein - a domain that only folds when attached to a neighbouring folded domain. This multidomain protein folds in a very different manner compared to those formed from adjacent domains which can fold in isolation.

Dynamic force spectroscopy is rapidly becoming a standard biophysical technique. Significant advances in the methods of analysis of force data have resulted in ever more complex systems being studied. The use of cloning systems to produce homologous tandem repeats rather than the use of endogenous multidomain proteins has facilitated these developments. What is poorly addressed are the physical properties of these constructed polyproteins. Are the properties of the individual domains in the construct independent of one another or attenuated by adjacent domains? We present data for a construct of eight fibronectin type III domains from the human form of tenascin that exhibits approximately 1 kcal mol(-1) increase in stability compared to the monomer. This effect is salt and pH dependent, suggesting that the stabilization results from electrostatic interactions, possibly involving charged residues at the interfaces of the domains. Kinetic analysis shows that this stabilization reflects a slower unfolding rate. Clearly, if domain-domain interactions affect the unfolding force, this will have implications for the comparison of absolute forces between types of domains. Mutants of the tenascin 8-mer construct exhibit the same change in stability as that observed for the corresponding mutation in the monomer. And when Phi-values are calculated for the 8-mer construct, the pattern is similar to that observed for the monomer. Therefore, mutational analyses to resolve mechanical unfolding pathways appear valid. Importantly, we show that interactions between the domains may be masked by changes in experimental conditions.

Spectrin is an elongated structural protein which forms, through inter-subunit interaction, a two-dimensional lattice-work of support for the cytoskeleton and membrane of a cell. The head-to-head interaction of the α- and β subunits, the “tetramer interaction,” is of particular interest, as here two partially unstructured domains undergo a coupled binding and folding reaction to yield a new three-helix bundled domain. In mammals, spectrin tetramers are found in two isoforms, SpI and SpII. SpI, the erythrocyte spectrin, forms relatively weak tetramers that exhibit slow folding and unfolding kinetics, believed to contribute to the intrinsic flexibility and durability of the red blood cell. In contrast SpII, the non-erythrocyte spectrin, rapidly associates to forms tetramers three orders of magnitude more stable than SpI. Intriguingly, the choice of β-subunit isoform does not dramatically alter the properties of the tetramer, indicating that difference in α-subunit isoform confers the majority of increased stability and association rate. Previous work has attributed this effect to a single residue (G46 in SpIα versus R37 in SpIIα) linking the N-terminal partially structured tail to the fully folded first domain of the α-subunit. In order to investigate the importance of helical propensity and charge character to the stability and association kinetics of the spectrin tetramer, we have made a series of point mutants in SpIα and SpIIα. Our early data suggest that increasing the helical propensity of residue 46 in SpIα by mutation to alanine accelerates the association kinetics, but does not alter the thermodynamic stability of the complex, while mutation to arginine dramatically increases the stability and folding kinetics.