Folding Thermodynamics Research Articles

Statistical-Mechanics Analyses on Thermodynamics of Protein Folding Constructed by Privalov and Co-Workers.

Privalov and co-workers estimated the changes in hydration enthalpy and entropy upon ubiquitin unfolding and their temperature dependences denoted by ΔHhyd(T) and ΔShyd(T), respectively, from experimentally measured enthalpies and entropies of transfer of various model compounds from gaseous phase to water. We calculate ΔHhyd(T) and ΔShyd(T) for ubiquitin by our statistical-mechanics theory where molecular and atomistic models are employed for water and protein structure, respectively. ΔHhyd(T) and ΔShyd(T) calculated are in remarkably good agreement with those estimated by Privalov and co-workers. By examining relative magnitudes and signs of the changes in a variety of constituents of ΔHhyd(T) and ΔShyd(T), we confirm that the hydrophobic effect is an essential force driving a protein to fold. Detailed and comprehensive explanations are given for our claim that the prevailing views of the hydrophobic effect are not capable of elucidating its weakening at low temperatures, whereas our updated view is. We find out problematic points of the changes in enthalpy and entropy upon protein unfolding denoted by ΔH°(T) and ΔS°(T), respectively, which are measured using the differential scanning calorimetry at low pH, suggesting a theoretical method of calculating ΔH°(T) and ΔS°(T) at pH ∼ 7.

The ribosome lowers the entropic penalty of protein folding

Most proteins fold during biosynthesis on the ribosome1, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies2–10. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol−1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding.

Native Characterization of Noncanonical Nucleic Acid Thermodynamics via Programmable Dynamic DNA Chemistry.

Folding thermodynamics, quantitatively described using parameters such as ΔGfold°, ΔHfold°, and ΔSfold°, is essential for characterizing the stability and functionality of noncanonical nucleic acid structures but remains difficult to measure at the molecular level. Leveraging the programmability of dynamic deoxyribonucleic acid (DNA) chemistry, we introduce a DNA-based molecular tool capable of performing a free energy shift assay (FESA) that directly characterizes the thermodynamics of noncanonical DNA structures in their native environments. FESA operates by the rational design of a reference DNA probe that is energetically equivalent to a target noncanonical nucleic acid structure in a series of toehold-exchange reactions, yet is structurally incapable of folding. As a result, a free energy shift (ΔΔGrxn°) is observed when plotting the reaction yield against the free energy of each toehold-exchange. We mathematically demonstrated that ΔGfold°, ΔHfold°, and ΔSfold° of the analyte can be calculated based on ΔΔGrxn°. After validating FESA using six DNA hairpins by comparing the measured ΔGfold°, ΔHfold°, and ΔSfold° values against predictions made by NUPACK software, we adapted FESA to characterize noncanonical nucleic acid structures, encompassing DNA triplexes, G-quadruplexes, and aptamers. This adaptation enabled the successful characterization of the folding thermodynamics for these complex structures under various experimental conditions. The successful development of FESA marks a paradigm shift and a technical advancement in characterizing the thermodynamics of noncanonical DNA structures through molecular tools. It also opens new avenues for probing fundamental chemical and biophysical questions through the lens of molecular engineering and dynamic DNA chemistry.

Quantitative entropy-enthalpy compensation in intraprotein interactions from model compound data.

Many small globular proteins exist in only two states-the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability. We focus on one such interaction, that between amide and aromatic groups. We provide a statistically convincing case for quantitative, linear entropy-enthalpy compensation in forming aromatic-amide interactions using published model compound transfer-free energy data.

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

Electrostatics introduce a trade-off between mesophilic stability and adaptation in halophilic proteins.

Extremophile organisms have adapted to extreme physicochemical conditions. Halophilic organisms, in particular, survive at very high salt concentrations. To achieve this, they have engineered the surface of their proteins to increase the number of short, polar and acidic amino acids, while decreasing large, hydrophobic and basic residues. While these adaptations initially decrease protein stability in the absence of salt, they grant halophilic proteins remarkable stability in environments with extremely high salt concentrations, where non-adapted proteins unfold and aggregate. The molecular mechanisms by which halophilic proteins achieve this, however, are not yet clear. Here, we test the hypothesis that the halophilic amino acid composition destabilizes the surface of the protein, but in exchange improves the stability in the presence of salts. To do that, we have measured the folding thermodynamics of various protein variants with different degrees of halophilicity in the absence and presence of different salts, and at different pH values to tune the ionization state of the acidic amino acids. Our results show that halophilic amino acids decrease the stability of halophilic proteins under mesophilic conditions, but in exchange improve salt-induced stabilization and solubility. We also find that, in contrast to traditional assumptions, contributions arising from hydrophobic effect and preferential ion exclusion are more relevant for haloadaptation than electrostatics. Overall, our findings suggest a trade-off between folding thermodynamics and halophilic adaptation to optimize proteins for hypersaline environments.

Energy landscapes of homopolymeric RNAs revealed by deep unsupervised learning

Conformational dynamics of RNA plays important roles in a variety of cellular functions such as transcriptional regulation, catalysis, scaffolding, and sensing. Recently, RNAs with low-complexity sequences have been shown to phase separate and form condensate phases similar to lowcomplexity protein domains. The affinity for phase separation and the material characteristics of RNA condensates are strongly dependent on sequence composition and patterning. We hypothesize that differences in the affinities for RNA phase separation can be uncovered by studying sequence-dependent conformational dynamics of single RNA chains. To this end, we have employed atomistic simulations and deep dimensionality reduction techniques to map temperature-dependent conformational free energy landscapes for 20 base-long homopolymeric RNA sequences: poly(U), poly(G), poly(C), and poly(A). The energy landscapes of homopolymeric RNAs reveal a plethora of metastable states with qualitatively different populations stemming from differences in base chemistry. Through detailed analysis of base, phosphate, and sugar interactions, we show that experimentally observed temperature-driven shifts in metastable state populations align with experiments on RNA phase transitions. Specifically, we find that the thermodynamics of unfolding of homopolymeric RNA follows the poly(G) > poly(A) > poly(C) > poly(U) order of stability, mirroring the propensity of RNA to form condensates. To conclude, this work shows that at least for homopolymeric RNA sequences the single-chain conformational dynamics contains sufficient information for predicting and quantifying condensate forming affinities of RNAs. Thus, we anticipate that atomically detailed studies of temeprature -dependent energy landscapes of RNAs will be a useful guide for understanding the propensity of various RNA molecules to form condensates.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST.

The accurate modeling of energetic contributions to protein structure is a fundamental challenge in computational approaches to protein analysis and design. We describe a general computational method, EmCAST (empirical Cα stabilization), to score and optimize the sequence to the structure in proteins. The method relies on an empirical potential derived from the database of the Cα dihedral angle preferences for all possible four-residue sequences, using the data available in the Protein Data Bank. Our method produces stability predictions that naturally correlate one-to-one with the experimental results for solvent-exposed mutation sites. EmCAST predicted four mutations that increased the stability of a three-helix bundle, UBA(1), from 2.4 to 4.8 kcal/mol by optimizing residues in both helices and turns. For a set of eight variants, the predicted and experimental stabilizations correlate very well (R2 = 0.97) with a slope near 1 and with a 0.16 kcal/mol standard error for EmCAST predictions. Tests against literature data for the stability effects of surface-exposed mutations show that EmCAST outperforms the existing stability prediction methods. UBA(1) variants were crystallized to verify and analyze their structures at an atomic resolution. Thermodynamic and kinetic folding experiments were performed to determine the magnitude and mechanism of stabilization. Our method has the potential to enable the rapid, rational optimization of natural proteins, expand the analysis of the sequence/structure relationship, and supplement the existing protein design strategies.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Structural and Functional Classification of G-Quadruplex Families within the Human Genome.

G-quadruplexes (G4s) are short secondary DNA structures located throughout genomic DNA and transcribed RNA. Although G4 structures have been shown to form in vivo, no current search tools that examine these structures based on previously identified G-quadruplexes and filter them based on similar sequence, structure, and thermodynamic properties are known to exist. We present a framework for clustering G-quadruplex sequences into families using the CD-HIT, MeShClust, and DNACLUST methods along with a combination of Starcode and BLAST. Utilizing this framework to filter and annotate clusters, 95 families of G-quadruplex sequences were identified within the human genome. Profiles for each family were created using hidden Markov models to allow for the identification of additional family members and generate homology probability scores. The thermodynamic folding energy properties, functional annotation of genes associated with the sequences, scores from different prediction algorithms, and transcription factor binding motifs within a family were used to annotate and compare the diversity within and across clusters. The resulting set of G-quadruplex families can be used to further understand how different regions of the genome are regulated by factors targeting specific structures common to members of a specific cluster.

Mega-scale analysis of protein folding stability in biology and protein design.

Progress in DNA sequencing and machine learning have illuminated protein sequences and structures on an enormous scale, but protein thermodynamics remains invisible and largely unknown. New approaches are needed to reveal protein folding thermodynamics on the same enormous scale to overcome challenges in biology, medicine, and engineering. We present cDNA display proteolysis, a new method for measuring protein folding stability for up to 900,000 protein domains in a one-week experiment. From 1.7 million measurements in total, we curated a set of ∼800,000 high-quality folding stabilities comprehensively examining single amino acid variants of ∼350 natural and ∼250 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also demonstrated applications of our method and dataset to protein design. The unique scale, speed, and accuracy of cDNA display proteolysis hold the potential to reveal protein folding thermodynamics across the entire universe of protein domains.

Separating the thermodynamic impact of non-specific interfacial interactions from confinement effects on protein stability.

The intracellular milieu is incredibly complex, containing myriad degrees of spatial restriction and highly variable interfacial chemistry. It is well established that this environmental complexity impacts diffusive properties, stability, and functional rates of proteins. The relative thermodynamic contributions of confinement versus non-specific interfacial interactions (quinary interactions), however, remain poorly understood. We are using reverse micelles (RMs) as a tool to differentiate between these effects. RMs are spontaneously organizing nanopools of water stabilized by surfactants in bulk non-polar solvent. By using optimized surfactant mixtures, the native states of proteins can be maintained in the RM water core. We have executed folding studies on a wide variety of proteins using varied surfactant mixtures with uniform degrees of confinement. We investigate both equilibrium folding thermodynamics and folding kinetics using various spectroscopic approaches. Our findings begin to unveil the complex relationship between a protein's thermodynamic stability and its local solvating environment, helping to dissect the differential impacts of confinement and quinary interactions.

Metal-binding and folding thermodynamics of Escherichia coli ribonuclease HI related to its catalytic function

Escherichia coli ribonuclease HI (RNH) hydrolyzes the RNA strands of RNA/DNA hybrids in the presence of Mg2+ at the highest level, relative to other metal ions. The Mg2+ binding affinity was 8.39 × 103 M−1, which was lower than those of other metal ions. The low-affinity binder can express the maximum catalytic activity of RNH. The stability of RNH increased with increasing metal ion concentration, except for Zn2+. The thermodynamic origin for enhancing the stability of RNH with Mg2+ was more favorable entropy compared to those with other metal ions, indicating that Mg2+ binding changes the RNH structure while maintaining flexibility. Upon H124A mutation, the metal ion binding affinities decreased for Mn2+ and Zn2+ to a relatively large extent. The present thermodynamic analyses provide information on the structural dynamics of RNH with metal ion exchangeable binding, which can reasonably explain the metal-ion-dependent catalytic activity.

DrTransformer: Heuristic cotranscriptional RNA folding using the nearest neighbor energy model.

Folding during transcription can have an important influence on the structure and function of RNA molecules, as regions closer to the 5' end can fold into metastable structures before potentially stronger interactions with the 3' end become available. Thermodynamic RNA folding models are not suitable to predict structures that result from cotranscriptional folding, as they can only calculate properties of the equilibrium distribution. Other software packages that simulate the kinetic process of RNA folding during transcription exist, but they are mostly applicable for short sequences. We present a new algorithm that tracks changes to the RNA secondary structure ensemble during transcription. At every transcription step, new representative local minima are identified, a neighborhood relation is defined and transition rates are estimated for kinetic simulations. After every simulation, a part of the ensemble is removed and the remainder is used to search for new representative structures. The presented algorithm is deterministic (up to numeric instabilities of simulations), fast (in comparison with existing methods), and it is capable of folding RNAs much longer than 200 nucleotides. This software is open-source and available at https://github.com/ViennaRNA/drtransformer. Supplementary data are available at Bioinformatics online.

A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity. This reduced hydrophobic effect is probably caused by water ordering due to the small number of hydration shells between the cavity and protein substrate surfaces. Hence, encapsulated protein substrates can undergo a process similar to cold denaturation in which unfolding is promoted by ordered water molecules. Our findings are likely to be relevant to encapsulated substrates in chaperonin systems, in general, and are consistent with the iterative annealing mechanism of action proposed for GroEL/ES.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

Effects of Familial Alzheimer's Disease Mutations on the Folding Free Energy and Dipole-Dipole Interactions of the Amyloid β-Peptide.

Familial Alzheimer's disease (FAD) mutations of the amyloid β-peptide (Aβ) are known to lead to early onset and more aggressive Alzheimer's disease. FAD mutations such as "Iowa" (D23N), "Arctic" (E22G), "Italian" (E22K), and "Dutch" (E22Q) have been shown to accelerate Aβ aggregation relative to the wild-type (WT). The mechanism by which these mutations facilitate increased aggregation is unknown, but each mutation results in a change in the net charge of the peptide. Previous studies have used nonpolarizable force fields to study Aβ, providing some insight into how this protein unfolds. However, nonpolarizable force fields have fixed charges that lack the ability to redistribute in response to changes in local electric fields. Here, we performed polarizable molecular dynamics simulations on the full-length Aβ42 of WT and FAD mutations and calculated folding free energies of the Aβ15-27 fragment via umbrella sampling. By studying both the full-length Aβ42 and a fragment containing mutations and the central hydrophobic cluster (residues 17-21), we were able to systematically study how these FAD mutations impact secondary and tertiary structure and the thermodynamics of folding. Electrostatic interactions, including those between permanent and induced dipoles, affected side-chain properties, salt bridges, and solvent interactions. The FAD mutations resulted in shifts in the electronic structure and solvent accessibility at the central hydrophobic cluster and the hydrophobic C-terminal region. Using umbrella sampling, we found that the folding of the WT and E22 mutants is enthalpically driven, whereas the D23N mutant is entropically driven, arising from a different unfolding pathway and peptide-bond dipole response. Together, the unbiased, full-length, and umbrella sampling simulations of fragments reveal that the FAD mutations perturb nearby residues and others in hydrophobic regions to potentially alter solubility. These results highlight the role electronic polarizability plays in amyloid misfolding and the role of heterogeneous microenvironments that arise as conformational change takes place.

Analysis of the effect of 1-Allyl-3-Methylimidazolium chloride on thermodynamic stability, folding kinetics, and motional dynamics of horse cytochrome c

1-allyl-3-methylimidazolium chloride (AMIMCl) acts as a potential green solvent for proteins. The present work provides a possible pathway by which the structural, kinetic, thermodynamic, and folding properties of horse cytochrome c (cyt c) are affected in green aqueous-AMIMCl systems. Analysis of the effect of AMIMCl on thermodynamic stability, refolding/unfolding kinetics, and motional dynamics of cyt c provided important information, (i) AMIMCl decreases the thermodynamic stability of reduced cyt c and also strengthens the guanidinium chloride (GdmCl)-mediated decrease in thermodynamic stability of protein, (ii) AMIMCl reduces the thermal-fluctuation of Met80-containing omega-loop of natively-folded compact state of carbonmonoxycytochrome c (MCO-state) due to polyfunctional interactions between the AMIM+ and different groups of protein, (iii) AMIMCl shifts the kinetic chevron plot, ln kobs[GdmCl] to the lower concentration of GdmCl, (iv) AMIMCl shifts the refolding and unfolding limps to vertically downwards and upwards, respectively, and (v) AMIMCl reducing the unfolding free energy estimated by both thermodynamic and kinetic analysis.