Folding Thermodynamics Research Articles

Role of Cys residues of C-terminal SH2 domain of phosphoinositide 3-kinase in its conformational stability and CD28-binding ability

Proteins fold into their most stable yet dynamic structures that enable their functions and exist in a balance between stability and instability. Protein folding and binding can be evaluated based on their thermodynamics determined using various methods. In this study, we analyzed the folding and binding thermodynamics of the C-terminal Src homology 2 domain (cSH2) of phosphoinositide 3-kinase and its Cys mutants, C656S, C659V, C670L, and C656S/C659V/C670L. The protein binds to the consensus sequence including phosphorylated Tyr, and the binding affinity to CD28-derived phosphopeptides decreased upon the Cys mutation: wild-type > C656S > C659V > C670L > C656S/C659V/C670L. The decreased affinity was mainly due to the increased binding enthalpy change. The thermal stability analysis showed the opposite tendency: wild-type < C656S < C659V < C670L < C656S/C659V/C670L, indicating a trade-off in protein architecture between stability and binding function. Based on the crystal structures of the cSH2 wild-type and C656S/C659V/C670L, the increased stability could be explained by the increased hydrophobic contacts. The wild-type protein would have evolved to have a native structure while maintaining the dynamics to express its binding function. Furthermore, a unique unfolding transition was observed for the cSH2 wild-type, possibly due to Cys oxidation, which was significantly changed upon the single mutation of Cys residues. We also demonstrated the validity of various methods for analyzing protein stability.

Water's Unusual Thermodynamics in the Realm of Physical Chemistry.

While it is known since the early work by Edsall, Frank and Evans, Kauzmann, and others that the thermodynamics of solvation of nonpolar solutes in water is unusual and has implications for the thermodynamics of protein folding, only recently have its connections with the unusual temperature dependence of the density of solvent water been illuminated. Such density behavior is, in turn, one of the manifestations of a nonstandard thermodynamic pattern contemplating a second, liquid-liquid critical point at conditions of temperature and pressure at which water exists as a deeply supercooled liquid. Recent experimental and computational work unambiguously points toward the existence of such a critical point, thereby providing concrete answers to the questions posed by the 1976 pioneering experiments by Speedy and Angell and the associated "liquid-liquid transition hypothesis" posited in 1992 by Stanley and co-workers. Challenges of this phenomenology to the branch of Statistical Mechanics remain.

Crowding-induced protein destabilization in the absence of soft attractions

It is generally assumed that volume exclusion by macromolecular crowders universally stabilizes the native states of proteins and destabilization suggests soft attractions between crowders and protein. Here we show that proteins can be destabilized even by crowders that are purely repulsive. With a coarse-grained sequence-based model, we study the folding thermodynamics of two sequences with different native folds, a helical hairpin and a β-barrel, in a range of crowder volume fractions, φc. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with φc. Hence, for all φc>0, U is entropically disfavored relative to N. This entropy-centric view holds for the helical hairpin protein, which is stabilized under all crowded conditions as quantified by changes in either the folding midpoint temperature, Tm, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-φc conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at T<Tm, such that U is only weakly disfavored entropically by the crowders; and 2) a transient, compact, and relatively low-energy nonnative state that has a maximum population of only a few percent at φc=0, but increasing monotonically with φc. Overall, protein destabilization driven by hard-core effects appears possible when a compaction of U leads to even a modest population of compact nonnative states that are energetically competitive with N.

Characterization of early and late transition states of the folding pathway of a SH2 domain.

Albeit SH2 domains are abundant protein–protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C‐terminal SH2 domain of SHP2, conducted through a combination of site‐directed mutagenesis and kinetic (un)folding experiments (Φ‐value analysis). The energetic profile of the folding reaction of the C‐SH2 domain is described by a three‐state mechanism characterized by the presence of two transition states and a high‐energy intermediate. The production of 29 site‐directed variants allowed us to calculate the degree of native‐like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ‐values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N‐terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.

Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5′GGUC3′ paired to 3′CUGG5′ separately from other 5′GU3′/3′UG5′ stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.

Interdomain interactions in Grb2 revealed by the conformational stability and CD28 binding analysis

Growth-factor receptor-bound protein 2 (Grb2) is an adaptor protein involved in signal transduction. The protein contains a central SH2 domain with N-terminal and C-terminal SH3 domains. We analyzed the SH3 involvement in Grb2 binding to the cytoplasmic region of CD28 receptor, using Grb2 and its SH3-deletion mutants, Grb2_nSH3del and Grb2_cSH3del, as the monomer state. The CD28 binding affinity of Grb2_nSH3del determined from surface plasmon resonance experiments was similar to that of Grb2_cSH3del, which was lower than that of Grb2 and higher than that of Grb2 SH2. The findings indicated that one of the SH3 domains is involved in CD28 binding. We also analyzed the thermal stabilities of Grb2 and its SH3-deletion mutants using differential scanning calorimetry, showing that the order of stability was Grb2_nSH3del < Grb2 < Grb2_cSH3del. Folding thermodynamics results indicated that SH3 domains, particularly nSH3, contribute to stabilizing the structure of Grb2, possibly due to the interdomain interaction.

Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (NaV) subtypes, including NaV1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at NaV1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV1.4≈1.2 > 1.7, while the native isomer inhibited NaV1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant NaV subtypes.

Lysine-Dependent Entropy Effects in the B. subtilis Lysine Riboswitch: Insights from Single-Molecule Thermodynamic Studies.

Riboswitches play an important role in RNA-based sensing/gene regulation control for many bacteria. In particular, the accessibility of multiple conformational states at physiological temperatures allows riboswitches to selectively bind a cognate ligand in the aptamer domain, which triggers secondary structural changes in the expression platform, and thereby "switching" between on or off transcriptional or translational states for the downstream RNA. The present work exploits temperature-controlled, single-molecule total internal reflection fluorescence (TIRF) microscopy to study the thermodynamic landscape of such ligand binding/folding processes, specifically for the Bacillus subtilis lysine riboswitch. The results confirm that the riboswitch folds via an induced-fit (IF) mechanism, in which cognate lysine ligand first binds to the riboswitch before structural rearrangement takes place. The transition state to folding is found to be enthalpically favored (ΔHfold‡ < 0), yet with a free-energy barrier that is predominantly entropic (-TΔSfold‡ > 0), which results in folding (unfolding) rate constants strongly dependent (independent) of lysine concentration. Analysis of the single-molecule kinetic "trajectories" reveals this rate constant dependence of kfold on lysine to be predominantly entropic in nature, with the additional lysine conferring preferential advantage to the folding process by the presence of ligands correctly oriented with respect to the riboswitch platform. By way of contrast, van't Hoff analysis reveals enthalpic contributions to the overall folding thermodynamics (ΔH0) to be surprisingly constant and robustly independent of lysine concentration. The results demonstrate the crucial role of hydrogen bonding between the ligand and riboswitch platform but with only a relatively modest fraction (45%) of the overall enthalpy change needed to access the transition state and initiate transcriptional switching.

Correction to: Thermodynamics of protein folding: methodology, data analysis and interpretation of data

Correction to: Thermodynamics of protein folding: methodology, data analysis and interpretation of data

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Predicting and Simulating Mutational Effects on Protein Folding Kinetics.

Mutational perturbations of protein structures, i.e., phi-value analysis, are commonly employed to probe the extent of involvement of a particular residue in the rate-determining step(s) of folding. This generally involves the measurement of folding thermodynamic parameters and kinetic rate constants for the wild-type and mutant proteins. While computational approaches have been reasonably successful in understanding and predicting the effect of mutations on folding thermodynamics, it has been challenging to explore the same on kinetics due to confounding structural, energetic, and dynamic factors. Accordingly, the frequent observation of fractional phi-values (mean of ~0.3) has resisted a precise and consistent interpretation. Here, we describe how to construct, parameterize, and employ a simple one-dimensional free energy surface model that is grounded in the basic tenets of the energy landscape theory to predict and simulate the effect of mutations on folding kinetics. As a proof of principle, we simulate one-dimensional free energy profiles of 806 mutations from 24 different proteins employing just the experimental destabilization as input, reproduce the relative unfolding activation free energies with a correlation of 0.91, and show that the mean phi-value of 0.3 essentially corresponds to the extent of stabilization energy gained at the barrier top while folding.

The effect of charged residue substitutions on the thermodynamics of protein-surface interactions.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

A reinforcement learning based method for protein’s differential scanning calorimetry signal separation

Differential scanning calorimetry (DSC) is a powerful technique to study protein stability, since the DSC test data provides valuable insights to characterize protein folding thermodynamics. Researchers in the drug discovery field need to manually analyze the DSC curves in multiple steps, such as baseline subtraction, data fitting, integration, and domain deconvolution. To improve the efficiency and consistency of data processing, machine learning methods for automatic DSC peak identification and baseline estimation were seen in prior research. However, the DSC’s automatic peak separation remained unexplored, despite its significant role in explaining the multi-domain protein unfolding. In this research, we propose a method based on reinforcement learning to separate the overlapping peaks of the DSC signal. We use two types of protein data to verify the effectiveness of this method. It automatically deconvolutes the peak signals into multiple sub-peaks. Our automated analysis method could lead to improved efficiency in DSC signal analysis when high volume data is involved. The code and data for this work can be found at: https://github.com/shuyu-wang/DSC_analysis_peak_separation.

Temperature and Guanidine Hydrochloride Effects on the Folding Thermodynamics of WW Domain and Variants.

We used simulations based on an all-atom Go model to calculate the folding temperatures (Tfs) and free energies (ΔGs) of two variants of the WW domain, which is a small all-β-sheet protein. The results, without adjusting any parameter, are in good agreement with experiments, thus validating the simulations. We then used the molecular transfer model to predict the changes in the ΔGs and Tfs as the guanidine hydrochloride concentration is varied. The predictions can be readily tested in experiments.

Vfold2D-MC: A Physics-Based Hybrid Model for Predicting RNA Secondary Structure Folding.

Accurate prediction of RNA structure and folding stability has a far-reaching impact on our understanding of RNA functions. Here we develop Vfold2D-MC, a new physics-based model, to predict RNA structure and folding thermodynamics from the sequence. The model employs virtual bond-based coarse-graining of RNA backbone conformation and generates RNA conformations through Monte Carlo sampling of the bond angles and torsional angles of the virtual bonds. Using a coarse-grained statistical potential derived from the known structures, we assign each conformation with a statistical weight. The weighted average over the conformational ensemble gives the entropy and free energy parameters for the hairpin, bulge, and internal loops, and multiway junctions. From the thermodynamic parameters, we predict RNA structures, melting curves, and structural changes from the sequence. Theory-experiment comparisons indicate that Vfold2D-MC not only gives improved structure predictions but also enables the interpretation of thermodynamic results for different RNA structures, including multibranched junctions. This new model sets a promising framework to treat more complicated RNA structures, such as pseudoknotted and intramolecular kissing loops, for which experimental thermodynamic parameters are often unavailable.

β-Hairpin Miniprotein Stabilization in Trehalose Glass Is Facilitated by an Emergent Compact Non-Native State.

From stem cell freeze-drying to organ storage, considerable recent efforts have been directed toward the development of new preservation technologies. A prominent protein stabilizing strategy involves vitrification in glassy matrices, most notably those formed of sugars such as the biologically relevant preservative trehalose. Here, we compare the folding thermodynamics of a model miniprotein in solution and in the glassy state of the sugars trehalose and glucose. Using synchrotron radiation circular dichroism (SRCD), we find that the same native structure persists in solution and glass. However, upon transition to the glass, a completely different, conformationally restricted unfolded state replaces the disordered denatured state found in solution, potentially inhibiting misfolding. Concomitantly, a large exothermic contribution is observed in glass, exposing the stabilizing effect of interactions with the sugar matrix on the native state. Our results shed light on the mechanism of protein stabilization in sugar glass and should aid in future preservation technologies.

Single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation

The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed in Escherichia coli in the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for “structural screening” of refolded proteins in the case that the “functional screening” like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.

The folding space of protein β2-microglobulin is modulated by a single disulfide bridge

Protein beta-2-microglobulin (β2m) is classically considered the causative agent of dialysis related amyloidosis, a conformational disorder that affects patients undergoing long-term hemodialysis. The wild type (WT) form, the ΔN6 structural variant, and the D76N mutant have been extensively used as model systems of β2m aggregation. In all of them, the native structure is stabilized by a disulfide bridge between the sulphur atoms of the cysteine residues 25 (at B strand) and 80 (at F strand), which has been considered fundamental in β2m fibrillogenesis. Here, we use extensive discrete molecular dynamics simulations of a full atomistic structure-based model to explore the role of this disulfide bridge as a modulator of the folding space of β2m. In particular, by considering different models for the disulfide bridge, we explore the thermodynamics of the folding transition, and the formation of intermediate states that may have the potential to trigger the aggregation cascade. Our results show that the dissulfide bridge affects folding transition and folding thermodynamics of the considered model systems, although to different extents. In particular, when the interaction between the sulphur atoms is stabilized relative to the other intramolecular interactions, or even locked (i.e. permanently established), the WT form populates an intermediate state featuring a well preserved core and two unstructured termini, which was previously detected only for the D76N mutant. The formation of this intermediate state may have important implications in our understanding of β2m fibrillogenesis.

Characteristic chemical probing patterns of loop motifs improve prediction accuracy of RNA secondary structures.

RNA structures play a fundamental role in nearly every aspect of cellular physiology and pathology. Gaining insights into the functions of RNA molecules requires accurate predictions of RNA secondary structures. However, the existing thermodynamic folding models remain less accurate than desired, even when chemical probing data, such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reactivities, are used as restraints. Unlike most SHAPE-directed algorithms that only consider SHAPE restraints for base pairing, we extract two-dimensional structural features encoded in SHAPE data and establish robust relationships between characteristic SHAPE patterns and loop motifs of various types (hairpin, internal, and bulge) and lengths (2–11 nucleotides). Such characteristic SHAPE patterns are closely related to the sugar pucker conformations of loop residues. Based on these patterns, we propose a computational method, SHAPELoop, which refines the predicted results of the existing methods, thereby further improving their prediction accuracy. In addition, SHAPELoop can provide information about local or global structural rearrangements (including pseudoknots) and help researchers to easily test their hypothesized secondary structures.