A Comprehensive Multidimensional-Embedded, One-Dimensional Reaction Coordinate for Protein Unfolding/Folding

The goal of the Dynameomics project is to perform, store, and analyze molecular dynamics simulations of representative proteins, of all known globular folds, in their native state and along their unfolding pathways. To analyze unfolding simulations, the location of the protein along the unfolding reaction coordinate (RXN) must be determined. Properties such as the fraction of native contacts and radius of gyration are often used; however, there is an issue regarding degeneracy with these properties, as native and nonnative species can overlap. Here, we used 15 physical properties of the protein to construct a multidimensional-embedded, one-dimensional RXN coordinate that faithfully captures the complex nature of unfolding. The unfolding RXN coordinates for 188 proteins (1534 simulations and 22.9 μs in explicit water) were calculated. Native, transition, intermediate, and denatured states were readily identified with the use of this RXN coordinate. A global native ensemble based on the native-state properties of the 188 proteins was created. This ensemble was shown to be effective for calculating RXN coordinates for folds outside the initial 188 targets. These RXN coordinates enable, high-throughput assignment of conformational states, which represents an important step in comparing protein properties across fold space as well as characterizing the unfolding of individual proteins.     

Effects of Topology and Sequence in Protein Folding Linked via Conformational Fluctuations

Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-β, whereas the folding of more local, all-α proteins typically exhibits a stronger sequence dependence. Here, we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3α, 4β + α, and β-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free-energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2000 sequence variants representing all possible hydrophobic or polar single- and double-point mutations. From an analysis of the subset of stability-neutral mutations, we find that folding is perturbed in a topology-dependent manner, with the β-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the β-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.     

How Does a Simplified-Sequence Protein Fold?

To investigate a putatively primordial protein we have simplified the sequence of a 56-residue α/β fold (the immunoglobulin-binding domain of protein G) by replacing it with polyalanine, polythreonine, and diglycine segments at regions of the sequence that in the folded structure are α-helical, β-strand, and turns, respectively. Remarkably, multiple folding and unfolding events are observed in a 15-μs molecular dynamics simulation at 330 K. The most stable state (populated at ∼20%) of the simplified-sequence variant of protein G has the same α/β topology as the wild-type but shows the characteristics of a molten globule, i.e., loose contacts among side chains and lack of a specific hydrophobic core. The unfolded state is heterogeneous and includes a variety of α/β topologies but also fully α-helical and fully β-sheet structures. Transitions within the denatured state are very fast, and the molten-globule state is reached in <1 μs by a framework mechanism of folding with multiple pathways. The native structure of the wild-type is more rigid than the molten-globule conformation of the simplified-sequence variant. The difference in structural stability and the very fast folding of the simplified protein suggest that evolution has enriched the primordial alphabet of amino acids mainly to optimize protein function by stabilization of a unique structure with specific tertiary interactions.     

Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for α/β proteins. Unfolding is initiated at the C-terminal strand (β_T) that lies at one edge of the β-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with β_T of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.     

Connecting Thermal and Mechanical Protein (Un)folding Landscapes

Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the force-induced unfolding behavior of filamin can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. This regime is typically explored by pulling experiments. While X may fail to resolve the TSE due to overlap with the unfolded ensemble just below the folding temperature, the overlap is minimal at lower temperatures where experiments are likely to be conducted. The TSE becomes increasingly structured with force, whereas the average order of structural events during unfolding remains roughly unchanged. The high-force regime is characterized by barrierless unfolding, and the unfolding time approaches a limit of ∼10 μs for the highest forces we studied. Finally, a combination of X and Q is shown to be a good reaction coordinate for almost the entire force range.     

Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (R_g) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The R_g of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where R_g decreased by ≈ 17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.     

Implementation of a k/k(0) method to identify long-range structure in transition states during conformational folding/unfolding of proteins

A previously introduced kinetic-rate constant (k/k(0)) method, where k and k(0) are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 A resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.     

Contribution of Long-Range Interactions to the Secondary Structure of an Unfolded Globin

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an α-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable α-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins.     

Ligand Entry into Fatty Acid Binding Protein via Local Unfolding Instead of Gap Widening

Fatty acid binding proteins play an important role in the transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still takes up fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer, and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like states, and two locally unfolded states in aqueous solution. Local unfolding of either βB–βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.     

A step-by-step classification algorithm of protein secondary structures based on double-layer SVM model

In this paper, a step-by-step classification algorithm based on double-layer SVM model is constructed to predict the secondary structure of proteins. The most important feature of this algorithm is to improve the prediction accuracy of α+β and α/β classes through transforming the prediction of two classes of proteins, α+β and α/β classes, with low accuracy in the past, into the prediction of all-α and all-β classes with high accuracy. A widely-used dataset, 25PDB dataset with sequence similarity lower than 40%, is used to evaluate this method. The results show that this method has good performance, and on the basis of ensuring the accuracy of other three structural classes of proteins, the accuracy of α+β class proteins is improved significantly.     

Union of Geometric Constraint-Based Simulations with Molecular Dynamics for Protein Structure Prediction

Although proteins are a fundamental unit in biology, the mechanism by which proteins fold into their native state is not well understood. In this work, we explore the assembly of secondary structure units via geometric constraint-based simulations and the effect of refinement of assembled structures using reservoir replica exchange molecular dynamics. Our approach uses two crucial features of these methods: i), geometric simulations speed up the search for nativelike topologies as there are no energy barriers to overcome; and ii), molecular dynamics identifies the low free energy structures and further refines these structures toward the actual native conformation. We use eight α-, β-, and α/β-proteins to test our method. The geometric simulations of our test set result in an average RMSD from native of 3.7 Å and this further reduces to 2.7 Å after refinement. We also explore the question of robustness of assembly for inaccurate (shifted and shortened) secondary structure. We find that the RMSD from native is highly dependent on the accuracy of secondary structure input, and even slightly shifting the location of secondary structure along the amino acid sequence can lead to a rapid decrease in RMSD to native due to incorrect packing.     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

Differential chemical and thermal unfolding pattern of Rv3588c and Rv1284 of Mycobacterium tuberculosis — A comparison by fluorescence and circular dichroism spectroscopy

The thermal and chemical unfolding pathways of two β carbonic anhydrases, Rv3588c and Rv1284 of Mycobacterium tuberculosis have been compared by fluorescence and circular dichroism. Chemical and thermal denaturation of the tertiary and secondary structures of these two ubiquitous enzymes of the pathogen reveals that the unfolding of Rv3588c is mediated through the formation of a molten globule intermediate with depleted tertiary structure. However, Rv1284 directly unfolds from the native to the unfolded state. Calculation of the thermodynamic parameters suggest that overall Rv3588c is more stable than Rv1284. Stern–Volmer analysis together with the fluorescence spectra of the proteins suggest that Trp115 in Rv1284 is more buried than Trp10 in Rv3588c. The tryptophan residues in both the proteins are surrounded by positively charged amino acid residues.     

Mapping Transient Partial Unfolding by Protein Engineering and Native-State Proteolysis

Transient partial unfolding of proteins under native conditions may have significant consequences in the biochemical and biophysical properties of proteins. Native-state proteolysis offers a facile way to investigate the thermodynamic and kinetic accessibilities of partially unfolded forms (cleavable forms) under native conditions. However, determination of the structure of the cleavable form, which is populated only transiently, remains challenging. Although in some cases partially cleaved products from proteolysis provide information on the structure of this elusive form, proteolysis of many proteins does not accumulate detectable intermediates. Here, we describe a systematic approach to determining structures of cleavable forms by protein engineering and native-state proteolysis. By devising φ_c analysis, which is analogous to conventional φ analysis, we have determined the structure of the cleavable form of Escherichia coli maltose-binding protein (MBP), which does not accumulate any partially cleaved products. We mutated 10 buried residues in MBP to alanine and determined φ_c values from the effects of the mutations on global stability and proteolytic susceptibility. The result of this analysis suggests that two C-terminal helices in MBP are unfolded in their cleavable form. The effect of ligand binding on proteolytic susceptibility and C-terminal deletion mutations also confirms the proposed structure. Our approach and methodology are generally applicable not only in elucidating the mechanism of proteolysis but also in investigating other important processes involving partial unfolding under native conditions such as protein misfolding and aggregation.     

NMR evidence for forming highly populated helical conformations in the partially folded hNck2 SH3 domain

Recent studies of several proteins implied that the folding of β-proteins may follow a nonhierarchical mechanism in which two major transitions are essential, i.e., the collapse of a random coil to form a nonnative helical intermediate, followed by a transformation into the native β-structure. We report that the first hNck2 SH3 domain, assuming an all-β barrel in the native form, can be reversibly transformed into a stable and nonnative helical state by acid-unfolding. We also conducted extensive NMR and mutagenesis studies that led to two striking findings: 1), NMR analysis reveals that in the helical state formed at pH 2.0, the first and last β-strands in the native form become unstructured, whereas the rest is surprisingly converted into two highly populated helices with a significantly limited backbone motion; and 2), a conserved four-residue sequence is identified on the second β-strand, a mutation of which suddenly renders the SH3 domain into a helical state even at pH 6.5, with NMR conformational and dynamic properties highly similar to those of the wild-type at pH 2.0. This observation implies that the region might contribute key interactions to disrupt the helical state, and to facilitate a further transformation into the native SH3 fold in the second transition.     

Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH^#), and activation entropy (ΔS^#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH^# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro¹¹⁴ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp¹¹⁵. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.     

Identification of Fibril-Like Tertiary Contacts in Soluble Monomeric α-Synuclein

Structural conversion of the presynaptic, intrinsically disordered protein α-synuclein into amyloid fibrils underlies neurotoxicity in Parkinson’s disease. The detailed mechanism by which this conversion occurs is largely unknown. Here, we identify a discrete pattern of transient tertiary interactions in monomeric α-synuclein involving amino acid residues that are, in the fibrillar state, part of β-strands. Importantly, this pattern of pairwise interactions does not correspond to that found in the amyloid state. A redistribution of this network of fibril-like contacts must precede aggregation into the amyloid structure.     

Protein thermostabilization by proline substitutions

Many recent approaches involving site-directed mutants have succeeded in increasing the thermostability of proteins. It is well known that replacements with proline residues reduce the conformational degrees of freedom in the main polypeptide chain and thus can increase protein thermostabilization. We have studied protein thermostabilization by introducing proline substitutions in the homologous oligo-1,6-glucosidases from various Bacillus strains which grow within different temperature ranges. As a consequence, the `proline rule' was proposed for protein thermostabilization. The principle of this rule is that an increase in the frequency of proline occurrence at β-turns and/or an increase in the total number of hydrophobic residues can enhance protein thermostability. We have generated several lines of evidence supporting the theory from the comparative analysis of oligo-1,6-glucosidases in their primary and secondary structures and molecular properties, the X-ray crystal structure analysis of the Bacillus cereus oligo-1,6-glucosidase, and the enhancement in thermostability of the oligo-1,6-glucosidase by cumulative replacements with prolines. As a new finding from the studies, two specific sites (second positions at β-turns and N1 positions of α-helices) were found to be the most critical to protein thermostabilization dependent on several structural prerequisites for proline substitution.     

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

We present a method for calculating the configurational-dependent diffusion coefficient of a globular protein as a function of the global folding process. Using a coarse-grained structure-based model, we determined the diffusion coefficient, in reaction coordinate space, as a function of the fraction of native contacts formed Q for the cold shock protein (TmCSP). We find nonmonotonic behavior for the diffusion coefficient, with high values for the folded and unfolded ensembles and a lower range of values in the transition state ensemble. We also characterized the folding landscape associated with an energetically frustrated variant of the model. We find that a low-level of frustration can actually stabilize the native ensemble and increase the associated diffusion coefficient. These findings can be understood from a mechanistic standpoint, in that the transition state ensemble has a more homogeneous structural content when frustration is present. Additionally, these findings are consistent with earlier calculations based on lattice models of protein folding and more recent single-molecule fluorescence measurements.