Designing cooperativity into the designed protein Top7

The topology of the designed protein Top7 is not found in natural proteins. Top7 shows signatures of non‐cooperative folding in both experimental studies and computer simulations. In particular, molecular dynamics of coarse‐grained structure‐based models of Top7 show a well‐populated C‐terminal folding‐intermediate. Since most similarly sized globular proteins are cooperative folders, the non‐natural topology of Top7 has been suggested as a reason for its non‐cooperative folding. Here, we computationally examine the folding of Top7 with the intent of making it cooperative. We find that its folding cooperativity can be increased in two ways: (a) Optimization of packing interactions in the N‐terminal half of the protein enables further folding of the C‐terminal intermediate. (b) Reduction in the packing density of the C‐terminal region destabilizes the intermediate. In practice, these strategies are implemented in our Top7 model through modifications to the contact‐map. These modifications do not alter the topology of Top7 but result in cooperative folding. Amino‐acid mutations that mimic these modifications also lead to a significant increase in folding cooperativity. Finally, we devise a method to randomize the sizes of amino‐acids within the same topology, and confirm that the structure of Top7 makes its folding sensitive to packing interactions. In contrast, the ribosomal protein S6, which has secondary structure similar to Top7, has folding which is much less sensitive to packing perturbations. Thus, it should be possible to make a sequence fold cooperatively to the structure of Top7, but to do so its side‐chain packing needs to be carefully designed. Proteins 2014; 82:364–374. © 2013 Wiley Periodicals, Inc.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Kinetically trapped metastable intermediate of a disulfide‐deficient mutant of the starch‐binding domain of glucoamylase

Refolding of a thermally unfolded disulfide‐deficient mutant of the starch‐binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and ¹H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half‐lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with β‐cyclodextrin as the native state, suggesting that the intermediate is highly‐ordered and native‐like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far‐UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off‐pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.     

Exploring the effects of sparse restraints on protein structure prediction

One of the main barriers to accurate computational protein structure prediction is searching the vast space of protein conformations. Distance restraints or inter‐residue contacts have been used to reduce this search space, easing the discovery of the correct folded state. It has been suggested that about 1 contact for every 12 residues may be sufficient to predict structure at fold level accuracy. Here, we use coarse‐grained structure‐based models in conjunction with molecular dynamics simulations to examine this empirical prediction. We generate sparse contact maps for 15 proteins of varying sequence lengths and topologies and find that given perfect secondary‐structural information, a small fraction of the native contact map (5%‐10%) suffices to fold proteins to their correct native states. We also find that different sparse maps are not equivalent and we make several observations about the type of maps that are successful at such structure prediction. Long range contacts are found to encode more information than shorter range ones, especially for α and αβ‐proteins. However, this distinction reduces for β‐proteins. Choosing contacts that are a consensus from successful maps gives predictive sparse maps as does choosing contacts that are well spread out over the protein structure. Additionally, the folding of proteins can also be used to choose predictive sparse maps. Overall, we conclude that structure‐based models can be used to understand the efficacy of structure‐prediction restraints and could, in future, be tuned to include specific force‐field interactions, secondary structure errors and noise in the sparse maps.     

Prediction of folding transition-state position (betaT) of small, two-state proteins from local secondary structure content

Folding kinetics of proteins is governed by the free energy and position of transition states. But attempts to predict the position of folding transition state on reaction pathway from protein structure have been met with only limited success, unlike the folding-rate prediction. Here, we find that the folding transition-state position is related to the secondary structure content of native two-state proteins. We present a simple method for predicting the transition-state position from their alpha-helix, turn and polyproline secondary structures. The method achieves 81% correlation with experiment over 24 small, two-state proteins, suggesting that the local secondary structure content, especially for content of alpha-helix, is a determinant of the solvent accessibility of the transition state ensemble and size of folding nucleus.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

Native Topology of the Designed Protein Top7 is Not Conducive to Cooperative Folding

Many single-domain proteins with <100 residues fold cooperatively; but the recently designed 92-residue Top7 protein exhibits clearly non-two-state behaviors. In apparent agreement with experiment, we found that coarse-grained, native-centric chain models, including potentials with and without elementary desolvation barriers, predicted that Top7 has a stable intermediate state in which the C-terminal fragment is folded while the rest of the chain remains disordered. We observed noncooperative folding in Top7 models that incorporated nonnative hydrophobic interactions as well. In contrast, free energy profiles deduced from models with desolvation barriers for a set of thirteen natural proteins with similar chain lengths and secondary structure elements suggested that they fold much more cooperatively than Top7. Buttressed by related studies on smaller natural proteins with chain lengths of ∼40 residues, our findings argue that the de novo native topology of Top7 likely imposed a significant restriction on the cooperativity achievable by any design for this target structure.     

Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs

B. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.     

Thermal unfolding simulations of a multimeric protein--transition state and unfolding pathways

The folding of an oligomeric protein poses an extra challenge to the folding problem because the protein not only has to fold correctly; it has to avoid nonproductive aggregation. We have carried out over 100 molecular dynamics simulations using an implicit solvation model at different temperatures to study the unfolding of one of the smallest known tetramers, p53 tetramerization domain (p53tet). We found that unfolding started with disruption of the native tetrameric hydrophobic core. The transition state for the tetramer to dimer transition was characterized as a diverse ensemble of different structures using Phi value analysis in quantitative agreement with experimental data. Despite the diversity, the ensemble was still native-like with common features such as partially exposed tetramer hydrophobic core and shifts in the dimer-dimer arrangements. After passing the transition state, the secondary and tertiary structures continued to unfold until the primary dimers broke free. The free dimer had little secondary structure left and the final free monomers were random-coil like. Both the transition states and the unfolding pathways from these trajectories were very diverse, in agreement with the new view of protein folding. The multiple simulations showed that the folding of p53tet is a mixture of the framework and nucleation-condensation mechanisms and the folding is coupled to the complex formation. We have also calculated the entropy and effective energy for the different states along the unfolding pathway and found that the tetramerization is stabilized by hydrophobic interactions.     

Analyses of the folding properties of ferredoxin‐like fold proteins by means of a coarse‐grained Gō model: Relationship between the free energy profiles and folding cores

The folding mechanisms of proteins with multi‐state transitions, the role of the intermediate states, and the precise mechanism how each transition occurs are significant on‐going research issues. In this study, we investigate ferredoxin‐like fold proteins which have a simple topology and multi‐state transitions. We analyze the folding processes by means of a coarse‐grained Gō model. We are able to reproduce the differences in the folding mechanisms between U1A, which has a high‐free‐energy intermediate state, and ADA2h and S6, which fold into the native structure through two‐state transitions. The folding pathways of U1A, ADA2h, S6, and the S6 circular permutant, S6_p54‐55, are reproduced and compared with experimental observations. We show that the ferredoxin‐like fold contains two common regions consisting folding cores as predicted in other studies and that U1A produces an intermediate state due to the distinct cooperative folding of each core. However, because one of the cores of S6 loses its cooperativity and the two cores of ADA2h are tightly coupled, these proteins fold into the native structure through a two‐state mechanism. Proteins 2014; 82:954–965. © 2013 Wiley Periodicals, Inc.     

Characterization of the unfolding pathway of the cell-cycle protein p13suc1 by molecular dynamics simulations: implications for domain swapping

BACKGROUND: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle. RESULTS: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition state, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta2 and beta4 remain associated. CONCLUSIONS: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta2-beta4 residual structure is surprising, because beta4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Folding and stability of helical bundle proteins from coarse‐grained models

We develop a coarse‐grained model where solvent is considered implicitly, electrostatics are included as short‐range interactions, and side‐chains are coarse‐grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side‐chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics. Proteins 2013; 81:1200–1211. © 2013 Wiley Periodicals, Inc.     

Relationship between chain collapse and secondary structure formation in a partially folded protein

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil‐globule transition)? To answer this question, we measured small‐angle X‐ray scattering for a series of β‐lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil‐globule transitions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 651–658, 2014.     

Molecular dynamics simulations of an engineered T4 lysozyme exclude helix to sheet transition,and provide insights into long distance,intra‐protein switchable motion

An engineered variant of T4 lysozyme serves as a model for studying induced remote conformational changes in a full protein context. The design involves a duplicated surface helix, flanked by two loops, that switches between two different conformations spanning about 20 Å. Molecular dynamics simulations of the engineered protein, up to 1 μs, rule out α‐helix to β‐sheet transitions within the duplicated helix as suggested by others. These simulations highlight how the use of different force fields can lead to radical differences in the structure of the protein. In addition, Markov state modeling and transition path theory were employed to map a 6.6 μs simulation for possible early intermediate states and to provide insights into the onset of the switching motion. The putative intermediates involve the folding of one helical turn in the C‐terminal loop through energy driven, sequential rearrangement of nearby salt bridges around the key residue Arg63. These results provide a first step towards understanding the energetics and dynamics of a rather complicated intra‐protein motion.     

PackHelix: a tool for helix-sheet packing during protein structure prediction

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α‐helices and/or β‐strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β‐sheet in a protein, there has been little progress on generating native‐like packings of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β‐sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ₂ motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. Proteins 2011; Published 2011 Wiley‐Liss, Inc.