Folding Rate Research Articles

Proteome-wide landscape of solubility limits in a bacterial cell

Proteins are prone to aggregate when expressed above their solubility limits. Aggregation may occur rapidly, potentially as early as proteins emerge from the ribosome, or slowly, following synthesis. However, in vivo data on aggregation rates are scarce. Here, we classified the Escherichia coli proteome into rapidly and slowly aggregating proteins using an in vivo image-based screen coupled with machine learning. We find that the majority (70%) of cytosolic proteins that become insoluble upon overexpression have relatively low rates of aggregation and are unlikely to aggregate co-translationally. Remarkably, such proteins exhibit higher folding rates compared to rapidly aggregating proteins, potentially implying that they aggregate after reaching their folded states. Furthermore, we find that a substantial fraction (~ 35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our study provides a global view of aggregation rates and hence solubility limits of proteins in a bacterial cell.

Covalent Capture of a Collagen Mimetic Peptide with an Integrin-Binding Motif.

Collagen mimetic peptides (CMPs) are an excellent model to study the structural and biological properties of the extracellular matrix (ECM) due to ease of synthesis and variability in sequence. To ensure that synthetic materials accurately mimic the structure and function of natural collagen in the ECM, it is necessary to conserve the triple helix. However, CMP folding is subject to equilibrium, and frequently peptides exist in solution as both monomer and triple helix. Additionally, the stability of CMPs is highly dependent on peptide length and amino acid composition, leading to suboptimal performance. Here, we report the utility of covalent capture, a method to (a) direct the folding of a supramolecular triple helix and (b) form isopeptide bonds between the helix strands, in the design of an integrin-binding peptide with a GFOGER motif. Covalent capture effectively locked the triple helix and yielded a peptide with high thermal stability and a rapid folding rate. Compared to supramolecular triple helices bearing the same GFOGER-binding site, cell adhesion was substantially increased. In vitro assays using EDTA/Mg2+ and an anti-α2β1 antibody demonstrated the preservation of the high specificity of the binding event. This covalently captured integrin-binding peptide provides a template for the future design of bioactive ECM mimics, which can overcome limitations of supramolecular approaches for potential drug and biomaterial designs.

The protein folding rate and the geometry and topology of the native state

Proteins fold in 3-dimensional conformations which are important for their function. Characterizing the global conformation of proteins rigorously and separating secondary structure effects from topological effects is a challenge. New developments in applied knot theory allow to characterize the topological characteristics of proteins (knotted or not). By analyzing a small set of two-state and multi-state proteins with no knots or slipknots, our results show that 95.4% of the analyzed proteins have non-trivial topological characteristics, as reflected by the second Vassiliev measure, and that the logarithm of the experimental protein folding rate depends on both the local geometry and the topology of the protein’s native state.

Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A.

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔGu°'(H2O), of UBA(1) is 2.4 kcal mol-1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, kf, in the absence of a denaturant of 13,000 s-1 and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.

Changing the vectorial synthesis of HaloTag through circular permutation modulates aggregation propensity in cells

Though in vitro studies have produced detailed insight into mechanisms underlying protein folding, determining how cellular factors modulate folding remains challenging. Proteins have the opportunity to fold when synthesized from N- to C-terminus by the ribosome, which builds polypeptide chains at a rate of around 10 residues per second in E. coli cells. This is much slower than the average folding rate of most small proteins, giving the nascent chain time to sample various conformations during translation.

Mechanics analysis and structural design of a truss deployable antenna mechanism based on 3RR-3URU tetrahedral unit

The truss deployable antenna mechanism based on tetrahedral unit belongs to spatial multi closed-loop coupled overconstrained mechanism, which has the advantages of high stiffness, strong expansibility and high folding rate. The mechanical and vibration characteristics of a tetrahedral deployable unit and its combined unit mechanism based on 3RR-3URU are studied. Firstly, the idea of equivalent stiffness is applied to the force analysis of the motion process of tetrahedral unit mechanism, and the relationship among motion, actuation stiffness and actuation force is revealed by using Lagrange method and displacement coordination relationship. Secondly, the vibration simulation analysis of the combined unit based on tetrahedral unit is carried out by using ADAMS vibration analysis module, the first 20 modes and natural frequencies are solved. In addition, the harmonic response is analyzed to obtain the sensitive frequency that the mechanism should avoid. Finally, the program modeling and detailed structure design of tetrahedral truss deployable antenna mechanism are investigated. Furthermore, experiments are carried out based on the developed combined unit prototype and one circle deployable mechanism prototype. The research results provide a reference for the structural optimization and engineering application of this kind of deployable antenna mechanism.

Current structure predictors are not learning the physics of protein folding.

Summary Motivation. Predicting the native state of a protein has long been considered a gateway problem for understanding protein folding. Recent advances in structural modeling driven by deep learning have achieved unprecedented success at predicting a protein’s crystal structure, but it is not clear if these models are learning the physics of how proteins dynamically fold into their equilibrium structure or are just accurate knowledge-based predictors of the final state. Results. In this work, we compare the pathways generated by state-of-the-art protein structure prediction methods to experimental data about protein folding pathways. The methods considered were AlphaFold 2, RoseTTAFold, trRosetta, RaptorX, DMPfold, EVfold, SAINT2 and Rosetta. We find evidence that their simulated dynamics capture some information about the folding pathway, but their predictive ability is worse than a trivial classifier using sequence-agnostic features like chain length. The folding trajectories produced are also uncorrelated with experimental observables such as intermediate structures and the folding rate constant. These results suggest that recent advances in structure prediction do not yet provide an enhanced understanding of protein folding. Availability. The data underlying this article are available in GitHub at https://github.com/oxpig/structure-vs-folding/Supplementary information Supplementary data are available at Bioinformatics online.

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a β-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1–2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the β-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

Calreticulin expression and localization in relation to exchangeable Ca2+ during pollen development in Petunia

BackgroundPollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development.ResultsUsing northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm.ConclusionOur present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.

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.

Efficient sampling of high-dimensional free energy landscapes using adaptive reinforced dynamics.

Enhanced sampling methods such as metadynamics and umbrella sampling have become essential tools for exploring the configuration space of molecules and materials. At the same time, they have long faced a number of issues such as the inefficiency when dealing with a large number of collective variables (CVs) or systems with high free energy barriers. Here we show that, with clustering and adaptive tuning techniques, the reinforced dynamics (RiD) scheme can be used to efficiently explore the configuration space and free energy landscapes with a large number of CVs or systems with high free energy barriers. We illustrate this by studying various representative and challenging examples. First we demonstrate the efficiency of adaptive RiD compared with other methods and construct the nine-dimensional (9D) free energy landscape of a peptoid trimer, which has energy barriers of more than 8 kcal mol-1. We then study the folding of the protein chignolin using 18 CVs. In this case, both the folding and unfolding rates are observed to be 4.30 μs-1. Finally, we propose a protein structure refinement protocol based on RiD. This protocol allows us to efficiently employ more than 100 CVs for exploring the landscape of protein structures and it gives rise to an overall improvement of 14.6 units over the initial global distance test-high accuracy (GDT-HA) score.

Native state of natural proteins optimizes local entropy.

The differing ability of polypeptide conformations to act as the native state of proteins has long been rationalized in terms of differing kinetic accessibility or thermodynamic stability. Building on the successful applications of physical concepts and sampling algorithms recently introduced in the study of disordered systems, in particular artificial neural networks, we quantitatively explore how well a quantity known as the local entropy describes the native state of model proteins. In lattice models and all-atom representations of proteins, we are able to efficiently sample high local entropy states and to provide a proof of concept of enhanced stability and folding rate. Our methods are based on simple and general statistical-mechanics arguments, and thus we expect that they are of very general use.

Mutational Analysis of Protein Folding Transition States: Phi Values.

The analysis of protein folding reactions by monitoring the kinetic effects of specifically designed single-point mutations, the so-termed phi-value analysis, has been a favorite technique to experimentally probe the mechanisms of protein folding. The idea behind phi-value analysis is that the effects that mutations have on the folding and unfolding rate constants report on the energetic/structural features of the folding transition state ensemble (TSE), which is the highest point in the free energy surface connecting the native and unfolded states, and thus the rate limiting step that ultimately defines the folding mechanism. For single-domain, two-state folding proteins, the general procedure to perform the phi-value analysis of protein folding is relatively simple to implement in the lab. Once the mutations have been produced and purified, the researcher needs to follow a few specific guidelines to perform the experiments and to analyze the data so produced. In this chapter, a step-by-step description of how to measure and interpret the effects induced by site-directed mutations on the folding and unfolding rate constants of a protein of interest is provided. Some possible solutions to the most typical problems that arise when performing phi-value analysis in the lab are also provided.

Engineered Metal-Binding Sites to Probe Protein Folding Transition States: Psi Analysis.

The formation of the transition state ensemble (TSE) represents the rate-limiting step in protein folding. The TSE is the least populated state on the pathway, and its characterization remains a challenge. Properties of the TSE can be inferred from the effects on folding and unfolding rates for various perturbations. A difficulty remains on how to translate these kinetic effects to structural properties of the TSE. Several factors can obscure the translation of point mutations in the frequently used method, "mutational Phi analysis." We take a complementary approach in "Psi analysis," employing rationally inserted metal binding sites designed to probe pairwise contacts in the TSE. These contacts can be confidently identified and used to construct structural models of the TSE. The method has been applied to multiple proteins and consistently produces a considerably more structured and native-like TSE than Phi analysis. This difference has significant implications to our understanding of protein folding mechanisms. Here we describe the application of the method and discuss how it can be used to study other conformational transitions such as binding.

Effects of Molecular Crowders on Single-Molecule Nucleic Acid Folding: Temperature-Dependent Studies Reveal True Crowding vs Enthalpic Interactions.

Biomolecular folding in cells can be strongly influenced by spatial overlap/excluded volume interactions (i.e., "crowding") with intracellular solutes. As a result, traditional in vitro experiments with dilute buffers may not accurately recapitulate biomolecule folding behavior in vivo. In order to account for such ubiquitous excluded volume effects, biologically inert polyethylene glycol (PEG) and polysaccharides (dextran and Ficoll) are often used as in vitro crowding agents to mimic in vivo crowding conditions, with a common observation that high concentrations of these polymers stabilize the more compact biomolecule conformation. However, such an analysis can be distorted by differences in polymer interactions with the folded vs unfolded conformers, requiring temperature-dependent analysis of the thermodynamics to reliably assess competing enthalpic vs entropic contributions and thus the explicit role of excluded volume. In this work, temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) is used to characterize the thermodynamic interaction between nucleic acids and common polymer crowders PEG, dextran, and Ficoll. The results reveal that PEG promotes secondary and tertiary nucleic acid folding by simultaneously increasing the folding rate while decreasing the unfolding rate, with temperature-dependent studies confirming that the source of PEG stabilization is predominantly entropic and consistent with a true excluded volume crowding mechanism. By way of contrast, neither dextran nor Ficoll induces any significant concentration-dependent change in nucleic acid folding stability at room temperature, but instead, stabilization effects gradually appear with a temperature increase. Such a thermal response indicates that both folding enthalpies and entropies are impacted by dextran and Ficoll. A detailed thermodynamic analysis of the kinetics suggests that, instead of true entropic molecular crowding, dextran and Ficoll associate preferentially with the unfolded vs folded nucleic acid conformer as a result of larger solvent accessible surface area, thereby skewing the free energy landscapes through both significant entropic/enthalpic contributions that compete and fortuitously cancel near room temperature.

Slowest-first protein translation scheme: Structural asymmetry and co-translational folding

Proteins are translated from the N to the C terminus, raising the basic question of how this innate directionality affects their evolution. To explore this question, we analyze 16,200 structures from the Protein Data Bank (PDB). We find remarkable enrichment of α helices at the C terminus and β strands at the N terminus. Furthermore, this α−β asymmetry correlates with sequence length and contact order, both determinants of folding rate, hinting at possible links to co-translational folding (CTF). Hence, we propose the “slowest-first” scheme, whereby protein sequences evolved structural asymmetry to accelerate CTF: the slowest of the cooperatively folding segments are positioned near the N terminus so they have more time to fold during translation. A phenomenological model predicts that CTF can be accelerated by asymmetry in folding rate, up to double the rate, when folding time is commensurate with translation time; analysis of the PDB predicts that structural asymmetry is indeed maximal in this regime. This correspondence is greater in prokaryotes, which generally require faster protein production. Altogether, this indicates that accelerating CTF is a substantial evolutionary force whose interplay with stability and functionality is encoded in secondary structure asymmetry.

Protein Folding Dynamics as Diffusion on a Free Energy Surface: Rate Equation Terms, Transition Paths, and Analysis of Single-Molecule Photon Trajectories.

The rates of protein (un)folding are often described as diffusion on the projection of a hyperdimensional energy landscape onto a few (ideally one) order parameters. Testing such an approximation by experiment requires resolving the reactive transition paths of individual molecules, which is now becoming feasible with advanced single-molecule spectroscopic techniques. This has also sparked the interest of theorists in better understanding reactive transition paths. Here we focus on these issues aiming to establish (i) practical guidelines for the mechanistic interpretation of transition path times (TPT) and (ii) methods to extract the free energy surface and protein dynamics from the maximum likelihood analysis of photon trajectories (MLA-PT). We represent the (un)folding rates as diffusion on a 1D free energy surface with the FRET efficiency as a reaction coordinate proxy. We then perform diffusive kinetic simulations on surfaces with two minima and a barrier, but with different shapes (curvatures, barrier height, and symmetry), coupled to stochastic simulations of photon emissions that reproduce current SM-FRET experiments. From the analysis of transition paths, we find that the TPT is inversely proportional to the barrier height (difference in free energy between minimum and barrier top) for any given surface shape, and that dividing the TPT into climb and descent segments provides key information about the barrier's symmetry. We also find that the original MLA-PT procedure used to determine the TPT from experiments underestimates its value, particularly for the cases with smaller barriers (e.g., fast folders), and we suggest a simple strategy to correct for this bias. Importantly, we also demonstrate that photon trajectories contain enough information to extract the 1D free energy surface's shape and dynamics (if TPT is >4-5-fold longer than the interphoton time) using the MLA-PT directly implemented with a diffusive free energy surface model. When dealing with real (unknown) experimental data, the comparison between the likelihoods of the free energy surface and discrete kinetic three-state models can be used to evaluate the statistical significance of the estimated free energy surface.

Identification of genotypic variants and its proteomic mutations of Brazilian SARS-CoV-2 isolates

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2′O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.

Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1–2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations.

Small Neutral Crowding Solute Effects on Protein Folding Thermodynamic Stability and Kinetics.

Molecular crowding is a ubiquitous phenomenon in biological systems, with significant consequences on protein folding and stability. Small compounds, such as the osmolyte trimethylamine N-oxide (TMAO), can also present similar effects. To analyze the effects arising from these solute-like molecules, we performed a series of crowded coarse-grained folding simulations. Two well-known proteins were chosen, CI2 and SH3, modeled by the alpha-carbon-structure-based model. In the simulations, the crowding molecules were represented by low-sized neutral atom beads in different concentrations. The results show that a low level of the volume fraction occupied by neutral agents can change protein stability and folding kinetics for the two systems. However, the kinetics were shown to be unaffected in their respective folding temperatures. The faster kinetics correlates with changes in the folding route for systems at the same temperature, where non-native contacts were shown to be relevant. The transition states of the two systems with and without crowders are similar. In the case of SH3, there are differences in the structuring of two strands, which may be associated with the increase in its folding rate, in addition to the destabilization of the denatured ensemble. The present study also detected a crossover in the thermodynamic stability behavior, previously observed experimentally and theoretically. As the temperature increases, crowders change from destabilizing to stabilizing agents.