Folding superfunnel to describe cooperative folding of interacting proteins

This paper proposes a generalization of the well‐known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein–protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein–protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009–1016. © 2016 Wiley Periodicals, Inc.     

Interplay between binding affinity and kinetics in protein–protein interactions

To clarify the interplay between the binding affinity and kinetics of protein–protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound‐state valley is deep with a barrier height of 12 ? 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920–933. © 2016 Wiley Periodicals, Inc.     

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.     

Coupling of folding and binding for unstructured proteins

There are now numerous examples of proteins that are unstructured or only partially structured under physiological conditions and yet are nevertheless functional. Such proteins are especially prevalent in eukaryotes. In many cases, intrinsically disordered proteins adopt folded structures upon binding to their biological targets. Many new examples of coupled folding and binding events have been reported recently, providing new insights into mechanisms of molecular recognition.     

Uncoupled binding and folding of immune signaling-related intrinsically disordered proteins

The classical protein structure-function paradigm has been challenged by the emergence of intrinsically disordered proteins (IDPs), the proteins that do not adopt well-defined three-dimensional structures under physiological conditions. This development was accompanied by the introduction of a “coupled binding and folding” paradigm that suggests folding of IDPs upon binding to their partners. However, our recent studies challenge this general view by revealing a novel, previously unrecognized phenomenon – uncoupled binding and folding. This biologically important mechanism is characteristic of members of a new family of IDPs involved in immune signaling and underlies their unusual properties including: (1) specific homodimerization, (2) the lack of folding upon binding to a well-folded protein, another IDP molecule, or to lipid bilayer membranes, and (3) the “scissors-cut paradox”. The third phenomenon occurs in diverse IDP interactions and suggests that properties of IDP fragments are not necessarily additive in the context of the entire protein. The “no disorder-to-order transition” type of binding is distinct from known IDP interactions and is characterized by an unprecedented observation of the lack of chemical shift and peak intensity changes in multidimensional NMR spectra, a fingerprint of proteins, upon complex formation. Here, I focus on those interactions of IDPs with diverse biological partners where the binding phase driven by electrostatic interactions is not be necessarily followed by the hydrophobic folding phase. I also review new multidisciplinary knowledge about immune signaling-related IDPs and show how it expands our understanding of cell function with multiple applications in biology and medicine.     

Protein folding

The problem of protein folding is that how proteins acquire their native unique three‐dimensional structure in the physiological milieu. To solve the problem, the following key questions should be answered: do proteins fold co‐ or post‐translationally, i.e. during or after biosynthesis, what is the mechanism of protein folding, and what is the explanation for fast folding of proteins? The two first questions are discussed in the current review. The general lines are to show that the opinion, that proteins fold after they are synthesized is hardly substantiated and suitable for solving the problem of protein folding and why proteins should fold cotranslationally. A possible tentative model for the mechanism of protein folding is also suggested. To this end, a thorough analysis is made of the biosynthesis, delivery to the folding compartments, and the rates of the biosynthesis, translocation and folding of proteins. A cursory attention is assigned to the role of GroEL/ES‐like chaperonins in protein folding.     

Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.     

The influence of intrinsic folding mechanism of an unfolded protein on the coupled folding-binding process during target recognition

Intrinsically disordered proteins (IDPs) are extensively involved in dynamic signaling processes which require a high association rate and a high dissociation rate for rapid binding/unbinding events and at the same time a sufficient high affinity for specific recognition. Although the coupled folding-binding processes of IDPs have been extensively studied, it is still impossible to predict whether an unfolded protein is suitable for molecular signaling via coupled folding-binding. In this work, we studied the interplay between intrinsic folding mechanisms and coupled folding-binding process for unfolded proteins through molecular dynamics simulations. We first studied the folding process of three representative IDPs with different folded structures, that is, c-Myb, AF9, and E3 rRNase. We found the folding free energy landscapes of IDPs are downhill or show low barriers. To further study the influence of intrinsic folding mechanism on the binding process, we modulated the folding mechanism of barnase via circular permutation and simulated the coupled folding-binding process between unfolded barnase permutant and folded barstar. Although folding of barnase was coupled to target binding, the binding kinetics was significantly affected by the intrinsic folding free energy barrier, where reducing the folding free energy barrier enhances binding rate up to two orders of magnitude. This accelerating effect is different from previous results which reflect the effect of structure flexibility on binding kinetics. Our results suggest that coupling the folding of an unfolded protein with no/low folding free energy barrier with its target binding may provide a way to achieve high specificity and rapid binding/unbinding kinetics simultaneously.     

Nonnative interactions in coupled folding and binding processes of intrinsically disordered proteins

Proteins function by interacting with other molecules, where both native and nonnative interactions play important roles. Native interactions contribute to the stability and specificity of a complex, whereas nonnative interactions mainly perturb the binding kinetics. For intrinsically disordered proteins (IDPs), which do not adopt rigid structures when being free in solution, the role of nonnative interactions may be more prominent in binding processes due to their high flexibilities. In this work, we investigated the effect of nonnative hydrophobic interactions on the coupled folding and binding processes of IDPs and its interplay with chain flexibility by conducting molecular dynamics simulations. Our results showed that the free-energy profiles became rugged, and intermediate states occurred when nonnative hydrophobic interactions were introduced. The binding rate was initially accelerated and subsequently dramatically decreased as the strength of the nonnative hydrophobic interactions increased. Both thermodynamic and kinetic analysis showed that disordered systems were more readily affected by nonnative interactions than ordered systems. Furthermore, it was demonstrated that the kinetic advantage of IDPs ("fly-casting" mechanism) was enhanced by nonnative hydrophobic interactions. The relationship between chain flexibility and protein aggregation is also discussed.     

A survey of flexible protein binding mechanisms and their transition states using native topology based energy landscapes

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interactions. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. Phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated Phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The Phi values for the formation of an antibody-antigen complex indicate a possible role for solvation of the interface in biomolecular association of large rigid proteins.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

Exploring the folding pathway of green fluorescent protein through disulfide engineering

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C‐termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m‐value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink‐induced perturbations in kinetics are useful for mapping the protein folding pathway.     

Chain length is the main determinant of the folding rate for proteins with three-state folding kinetics

We demonstrate that chain length is the main determinant of the folding rate for proteins with the three-state folding kinetics. The logarithm of their folding rate in water (k(f)) strongly anticorrelates with their chain length L (the correlation coefficient being -0.80). At the same time, the chain length has no correlation with the folding rate for two-state folding proteins (the correlation coefficient is -0.07). Another significant difference of these two groups of proteins is a strong anticorrelation between the folding rate and Baker's "relative contact order" for the two-state folders and the complete absence of such correlation for the three-state folders.     

The folding mechanics of a knotted protein

An increasing number of proteins are being discovered with a remarkable and somewhat surprising feature, a knot in their native structures. How the polypeptide chain is able to "knot" itself during the folding process to form these highly intricate protein topologies is not known. Here we perform a computational study on the 160-amino-acid homodimeric protein YibK, which, like other proteins in the SpoU family of MTases, contains a deep trefoil knot in its C-terminal region. In this study, we use a coarse-grained C(alpha)-chain representation and Langevin dynamics to study folding kinetics. We find that specific, attractive nonnative interactions are critical for knot formation. In the absence of these interactions, i.e., in an energetics driven entirely by native interactions, knot formation is exceedingly unlikely. Further, we find, in concert with recent experimental data on YibK, two parallel folding pathways that we attribute to an early and a late formation of the trefoil knot, respectively. For both pathways, knot formation occurs before dimerization. A bioinformatics analysis of the SpoU family of proteins reveals further that the critical nonnative interactions may originate from evolutionary conserved hydrophobic segments around the knotted region.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.     

Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Sub-microsecond protein folding

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.     

Assessing protein disorder and induced folding

Intrinsically disordered proteins (IDPs) defy the structure-function paradigm as they fulfill essential biological functions while lacking well-defined secondary and tertiary structures. Conformational and spectroscopic analyses showed that IDPs do not constitute a uniform family, and can be divided into subfamilies as a function of their residual structure content. Residual intramolecular interactions are thought to facilitate binding to a partner and then induced folding. Comprehensive information about experimental approaches to investigate structural disorder and induced folding is still scarce. We herein provide hints to readily recognize features typical of intrinsic disorder and review the principal techniques to assess structural disorder and induced folding. We describe their theoretical principles and discuss their respective advantages and limitations. Finally, we point out the necessity of using different approaches and show how information can be broadened by the use of multiples techniques.