Stability Of Globular Proteins Research Articles

Cover Image

Protein ScienceVolume 32, Issue 5 Cover ImageFree Access Cover Image First published: 17 April 2023 https://doi.org/10.1002/pro.4636AboutRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Crowders impact globular protein stability by their exclusion or inclusion from the vicinity of the protein. The degree of exclusion or inclusion is determined by the balance between hard and soft (chemical) interactions. The image depicts crowder and solvent molecules in the bulk and protein domains at equilibrium. Protein Science, 2023. https://doi.org/10.1002/pro.4573. Volume32, Issue5May 2023 RelatedInformation RecommendedImage SegmentationFundamentals of Digital Image Processing: A Practical Approach with Examples in Matlab, [1]Image SegmentationImage Processing: Principles and Applications, [1]Image SegmentationPractical Image and Video Processing Using MATLAB®, [1]Image Processing TechniquesJohn C. Russ, Encyclopedia of Imaging Science and Technology, [1]

Exploring the role of preferential solvation in the stability of globular proteins through the study of ovalbumin interaction with organic additives

<abstract> <p>The impact of denaturing and stabilizing osmolytes on protein conformational dynamics has been extensively explored due to the significant contribution of protein solvation to the stability, function, malfunction and regulation of globular proteins. We studied the effect of two nonspecific organic molecules, urea, which is a conventional denaturant, and dimethyl sulfoxide (DMSO), which is a multilateral organic solvent, on the stability and conformational dynamics of a non-inhibitory serpin, ovalbumin (OVA). A differential scanning microcalorimetry (DSC) experimental series conducted in the phosphate buffer solutions containing 0–30% of additives revealed the destabilizing impact of both urea and DMSO in a mild acidic media, manifested in the gradual decrease of thermal unfolding enthalpy and transition temperature. These findings differ from the results observed in our study of the mild alkaline DMSO buffered solutions of OVA, where the moderate stabilization of OVA was observed in presence of 5–10% of DMSO. However, the overall OVA interaction patterns with urea and DMSO are consistent with our previous findings on the stability and conformational flexibility of another model globular protein, α-chymotrypsin, in similar medium conditions. The obtained results could be explained by preferential solvation patterns. Positive preferential solvation of protein by urea in urea/water mixtures mainly weakens the hydrophobic interactions of the protein globule and eventually leads to the disruption of the tertiary structure within the whole range of urea concentrations. Alternatively, under certain experimental conditions in DMSO/water mixtures, positive preferential solvation by water molecules can be observed. We assume that the switch to the positive preferential solvation by DMSO, which is shown to have a soft maximum around 20–30% DMSO, could be shifted towards lower additive concentrations due to the intrinsic capability of ovalbumin OVA to convert into a heat-stable, yet flexible set of conformations that have increased the surface hydrophobicity, characteristic to molten-globule-like states.</p> </abstract>

Unfolded Lipase at Interfaces Studied via Interfacial Dilational Rheology: The Impact of Urea

Unfolding can interrupt the activity of enzymes. Lipase, the enzyme responsible for triglyceride catalysis, can be deactivated by unfolding, which can significantly affect the yield of enzymatic processes in biochemical engineering. Different agents can induce lipase unfolding, among which we study the impact of urea as a strong denaturant. Unfolding weakens the rigidity and stability of globular proteins, thereby changing the viscoelastic properties of the protein adsorbed layers. These changes can be detected and quantified using interfacial dilational rheology. The urea-induced unfolding of lipase destructs its globular structure, making it more flexible. The interfacial tension and viscoelastic moduli of lipase adsorbed layers reduce upon the addition of urea in the range of studied concentrations. The results agree with the theory that, upon unfolding, a distal region of the loop and tail domain forms adjacent to the proximal region of the interface. The exchange of matter between these regions reduces the viscoelasticity of the unfolded lipase adsorbed layers. Additionally, unfolding reduces the rigidity and brittleness of the lipase adsorbed layers: the aged adsorbed layer of native lipase can break upon high-amplitude perturbations of the interfacial area, unlike the case for urea-induced unfolded lipase.

Cation-π Interactions and Their Role in Assembling Collagen Triple Helices.

Cation-π interactions play a significant role in the stabilization of globular proteins. However, their role in collagen triple helices is less well understood and they have rarely been used in de novo designed collagen mimetic systems. In this study, we analyze the stabilizing and destabilizing effects in pairwise amino acid interactions between cationic and aromatic residues in both axial and lateral sequential relationships. Thermal unfolding experiments demonstrated that only axial pairs are stabilizing, while the lateral pairs are uniformly destabilizing. Molecular dynamics simulations show that pairs with an axial relationship can achieve a near-ideal interaction distance, but pairs in a lateral relationship do not. Arginine-π systems were found to be more stabilizing than lysine-π and histidine-π. Arginine-π interactions were then studied in more chemically diverse ABC-type heterotrimeric helices, where arginine-tyrosine pairs were found to form the best helix. This work helps elucidate the role of cation-π interactions in triple helices and illustrates their utility in designing collagen mimetic peptides.

A quantum chemical study on the hydration energetics of trimethylamine N-oxide, its protonated form and tert-butanol

Trimethylamine N-oxide (TMAO) stabilizes the native state of globular proteins and counteracts the destabilizing action of denaturants. However, at pH values lower than its pKa = 4.7, TMAO destabilizes the native state. We studied, via DFT calculations, the interactions of TMAO, its protonated form and isosteric tert-butanol with one, two and three water molecules in vacuo, PCM and SMD water. The energetic strength of the H-bonds the considered solutes make with three water molecules, coupled to a model of globular protein stability, allows us to propose a rationalization of the pH-dependent TMAO behavior toward globular proteins.

Some Clues about Enzymes from Psychrophilic Microorganisms.

Enzymes purified from psychrophilic microorganisms prove to be efficient catalysts at low temperatures and possess a great potential for biotechnological applications. The low-temperature catalytic activity has to come from specific structural fluctuations involving the active site region, however, the relationship between protein conformational stability and enzymatic activity is subtle. We provide a survey of the thermodynamic stability of globular proteins and their rationalization grounded in a theoretical approach devised by one of us. Furthermore, we provide a link between marginal conformational stability and protein flexibility grounded in the harmonic approximation of the vibrational degrees of freedom, emphasizing the occurrence of long-wavelength and excited vibrations in all globular proteins. Finally, we offer a close view of three enzymes: chloride-dependent α-amylase, citrate synthase, and β-galactosidase.

A simple model of protein cold denaturation

Cold denaturation is a general feature of the conformational stability of globular proteins. Its modelization is still an important goal of biophysics. We present a modified version of the Schellman-Brandts model that is able to reproduce the occurrence of two conformational transitions, cold and hot denaturations. The model suggests that: (a) globular protein cooperativity can be obtained by distinguishing an inner core, buried from water contact, in a spherical globule; (b) the latter is stabilized by the hydrophobic effect that can be modelled by the Gibbs free energy gain associated with the burial of nonpolar side chains in the inner core; (c) cold denaturation comes from the decrease in the stabilization provided by the hydrophobic effect at low temperature.

Investigation on the effect of nonpolar amino acids as macromolecular crowders on the stability of globular proteins

Addition of macromolecular crowders to biomolecules can mimic the interior of living cells, however, it is not always very evident whether the effect is general or dependent on the biomolecule-crowder pair specificity. In this study, we investigate the effect of nonpolar hydrophobic amino acids (Glycine, L-alanine, L-valine, L-leucine, L-isoleucine) as crowding agents on the structural and thermal stability of three proteins: human serum albumin (HSA), lysozyme (HEWL) and Ribonuclease-A (RNase-A) of varying α-helical content using temperature dependent circular dichroism (CD) spectroscopic technique. It has been observed that all these five amino acids do not substantially alter the secondary structure of proteins rather they stabilize their respective native forms. Thermal unfolding of each protein is found to be irreversible in nature with the extent of secondary structural loss during refolding being proportional to the α-helicity of the protein. Estimated thermodynamic parameters (van't Hoff enthalpy and heat capacity) show that enthalpic stabilization is protein specific and the added amino acids alter these parameters in different extent without following any specific trend. Our study affirms that conformational stability of proteins and the associated thermodynamic parameters do not necessarily bear a linear correlation with the hydrophobicity of amino acids; rather it is protein specific where the secondary structural content plays an important role.

General Counteraction Exerted by Sugars against Denaturants.

The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water.

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States.

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

The magnitude of macromolecular crowding caused by Dextran and Ficoll for the conformational stability of globular proteins

The polysaccharides Dextran and Ficoll are commonly used as crowding agents to mimic the cellular environment. They usually stabilize the native state of globular proteins, and this effect has been explained by the excluded volume idea: the reduction of the volume available to host the protein molecule shifts the equilibrium towards the folded conformation. However, the experimentally measured stabilization is lower than expected from theoretical models. We would like to show that, applying a classic scaled particle theory approach that considers the density of the solutions, we can provide reliable estimates of their stabilizing effect. Density is a fundamental requirement because it reflects the actual interactions among the solution molecules and allows the calculation of the number density and the volume packing density of the solution, two factors that play a major role in defining the magnitude of the solvent-excluded volume effect. Application of the model to aqueous solutions of glucose, sucrose, Dextran-70 and Ficoll-70 indicates that the stabilization effect of the two polysaccharides is similar to that of glucose and sucrose, and this happens due to the low number density of the polymer solutions.

Is water a good solvent for the denatured state of globular proteins?

Making extensive use of polymer chemistry concepts, it has recently been claimed that water is a good solvent for the denatured state of globular proteins [J. Mol. Biol. 432 (2020) 2882]. Here, I discuss how overinterpretation of classical polymer chemistry should be avoided and how recent ideas about the source of the hydrophobic effect can explain the role of water in the folding and conformational stability of globular proteins.

Thermodynamic and Evolutionary Coupling between the Native and Amyloid State of Globular Proteins

SummaryThe amyloid-like aggregation propensity present in most globular proteins is generally considered to be a secondary side effect resulting from the requirements of protein stability. Here, we demonstrate, however, that mutations in the globular and amyloid state are thermodynamically correlated rather than simply associated. In addition, we show that the standard genetic code couples this structural correlation into a tight evolutionary relationship. We illustrate the extent of this evolutionary entanglement of amyloid propensity and globular protein stability. Suppressing a 600-Ma-conserved amyloidogenic segment in the p53 core domain fold is structurally feasible but requires 7-bp substitutions to concomitantly introduce two aggregation-suppressing and three stabilizing amino acid mutations. We speculate that, rather than being a corollary of protein evolution, it is equally plausible that positive selection for amyloid structure could have been a driver for the emergence of globular protein structure.

Intrinsic disorder-based design of stable globular proteins.

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. In this work, we have successfully used algorithms for the evaluation of intrinsic disorder predisposition (such as PONDR® FIT and IsUnstruct) as tools for searching for the weakened regions in structured globular proteins. We have shown that the weakened regions found by these programs as regions with highest levels of predicted intrinsic disorder predisposition are a suitable target for introduction of stabilizing mutations.

Intrinsic Disorder-Based Design of Stable Globular Proteins.

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. This work summarizes our research on the effect of amino acid substitutions on the protein stability utilizing the outputs of the analysis of intrinsic disorder predisposition of target proteins. This allowed us to formulate the basis of one of the possible approaches to the stabilization of globular proteins. The idea is quite simple. To stabilize a protein as a whole, one needs to find its "weakest spot" and stabilize it, but the question is how this weak spot can be found in a query protein. Our approach is based on the utilization of the computational tools for the per-residue evaluation of intrinsic disorder predisposition to search for the "weakest spot" of a query protein (i.e., the region(s) with the highest local predisposition for intrinsic disorder). When such "weakest spot" is found, it can be stabilized through a limited number of point mutations by introducing order-promoting residues at hot spots, thereby increasing structural stability of a protein as a whole. Using this approach, we were able to obtain stable mutant forms of several globular proteins, such as Gαo, GFP, ribosome protein L1, and circular permutant of apical domain of GroEL.

Insight into the effect of glycerol on stability of globular proteins in high protein model system

A model system was established to investigate the effect of glycerol on stability of globular proteins. To study whether protective effect of glycerol is dependent on the nature of protein, four kinds of globular protein were used including α-lactalbumin, β-lactoglobulin, native lactoferrin and lysozyme. Differential scanning calorimetry (DSC) was used to study the thermal stability of the proteins with glycerol aqueous (0–80% v/v). The results showed that the stability of β-lactoglobulin and lysozyme (hydrophobicity-dominated proteins) increased with glycerol concentration. However, that of α-lactalbumin and lactoferrin (metalloproteins), regardless of the ion-binding state, initially increased and then decreased. Different protective effects were observed beyond 50% (v/v) glycerol concentrations in liquid phase. The secondary structure and tryptophan exposure remained unchanged. In conclusion, the protection of glycerol on protein stability was highly dependent on the nature of proteins.

Protein folding and quinary interactions: creating cellular organisation through functional disorder.

The marginal stability of globular proteins in the cell is determined by the balance between excluded volume effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organisation and known to have stabilising or destabilising effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilisation can be sufficient to produce functional partially unfolded states of globular proteins. The biological relevance of this mechanism is explored, involving intracellular phase separation and regulatory stress response mechanisms.

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein, an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.

Effect of heavy water on the conformational stability of globular proteins.

It is well established from the experimental point of view that the native state of globular proteins is more stable in heavy water than in water. No robust explanation, however, has been provided up to now. The application of the theoretical approach, originally devised to rationalize the general occurrence of cold denaturation, indicates that the magnitude of the solvent-excluded volume effect is slightly smaller in heavy water than in water and cannot explain the observed protein stabilization. The latter has to be due to the strength of protein-water van der Waals attractions which are weaker in heavy water due to the smaller molecular polarizability of D2 O compared with that of H2 O molecules. Since protein-water van der Waals attractions occur more in the denatured than in the native state, this contribution leads to a stabilization of the latter through a destabilization of the former.

Analysis and prediction of protein folding energy changes upon mutation by element specific persistent homology.

Site directed mutagenesis is widely used to understand the structure and function of biomolecules. Computational prediction of mutation impacts on protein stability offers a fast, economical and potentially accurate alternative to laboratory mutagenesis. Most existing methods rely on geometric descriptions, this work introduces a topology based approach to provide an entirely new representation of mutation induced protein stability changes that could not be obtained from conventional techniques. Topology based mutation predictor (T-MP) is introduced to dramatically reduce the geometric complexity and number of degrees of freedom of proteins, while element specific persistent homology is proposed to retain essential biological information. The present approach is found to outperform other existing methods in the predictions of globular protein stability changes upon mutation. A Pearson correlation coefficient of 0.82 with an RMSE of 0.92 kcal/mol is obtained on a test set of 350 mutation samples. For the prediction of membrane protein stability changes upon mutation, the proposed topological approach has a 84% higher Pearson correlation coefficient than the current state-of-the-art empirical methods, achieving a Pearson correlation of 0.57 and an RMSE of 1.09 kcal/mol in a 5-fold cross validation on a set of 223 membrane protein mutation samples. http://weilab.math.msu.edu/TML/TML-MP/. wei@math.msu.edu. Supplementary data are available at Bioinformatics online.