Heat-induced Conformational Changes Research Articles

Regioselective analysis of heat-induced conformational changes of β-lactoglobulin by quantitative liquid chromatography–mass spectrometry analysis of chemical labeling kinetics

β-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native β-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25–75 °C) and complete transitions (80–90 °C). Regioselective conformational changes of BLG-A in the native state (25 °C), sequential (70 °C) and complete transition (90 °C) were determined by quantitative liquid chromatography–mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 (both N-terminal chain), K60 (β-sheet C), K75 (β-sheet D), K77 (DE loop), K83 (β-sheet E), K100 and K101 (FG loop). The residues K14 (β-sheet A1), K47 (β-sheet B), K69, K70 (both β-sheet D), and K91 (β-sheet F) were not involved in conformational changes.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Laser Heating Nanoelectrospray Emitters for Fast Protein Melting Measurements with Mass Spectrometry.

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 μm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.

Effects of pressurized thermal processing on native proteins of raw skim milk and its concentrate

Heating, pressurization, and shearing can modify native milk proteins. The effects of pressurized heating (0.5 vs. 10 MPa at 75 or 95°C) with shearing (1,000 s-1) on proteins of raw bovine skim milk (SM, ∼9% total solids) and concentrated raw skim milk (CSM, ∼22% total solids) was investigated. The effects of evaporative concentration at 55°C and pressurized shearing (10 MPa, 1,000 s-1) at 20°C were also examined. Evaporative concentration of SM resulted in destabilization of casein micelles and dissociation of αS1- and β-casein, rendering CSM prone to further reactions. Treatment at 10 MPa and 1,000 s-1 at 20°C caused substantial dissociation of αS1- and β-casein in SM and CSM, with some dissociated caseins forming shear-induced soluble aggregates in CSM. The pressure applied at 10 MPa induced compression of the micelles and their dissociation in SM and CSM at 75 or 95°C, resulting in reduction of the micelle size. However, 10 MPa did not alter the mineral balance or whey proteins denaturation largely, except by reduction of some β-sheets and α-helices, due to heat-induced conformational changes at 75 and 95°C.

Process-Structure-Function in Association with the Main Bioactive of Black Rice Flour Sieving Fractions.

The aim of this work was to advance knowledge on the potential use of black rice different sieving fractions for various functional applications, through proximate analysis, thermal degradation kinetics of phytochemical and characterization of the thermal behavior of the main proteins, from the perspectives of their use as a food ingredient. The results indicated that the thermal degradation of phytochemicals followed a first-order reaction kinetics for all the tested fractions. The temperature-dependent degradation was adequately modeled according to the Arrhenius equation. The calculated activation energies (Ea) and k values were different among the four studied parameters. The kinetic parameters depended on the grinding and sieving degree, the anthocyanins being the most thermolabile compounds, thus affecting the antioxidant activity. Three protein fractions were identified by electrophoresis with different molecular weight, such as albumin, globulin, and glutelin. The fluorescence spectroscopy experiments revealed the sequential character of the heat-induced conformational changes, different molecular events being suggested, such as folding in the lower temperature range and unfolding at higher temperature. The significance of the study is evidenced by the need to identify and advance the process-structure-function relationships for various biologically active compounds from the perspective of obtaining food or ingredients nutritionally optimized.

The stability of triphasic oil-in-water Pickering emulsions can be improved by physical modification of hordein- and secalin-based submicron particles

The main objective of this work was to study the capability of either non-modified or physically-modified β-hordein and γ-secalin particles as Pickering emulsion stabilizers. A comparison was performed with zein-based particles as a commercial source of prolamins. Protein dispersions in ethanol-water medium were subjected to different heat treatments and then characterized. A simple anti-solvent precipitation method in combination with sonication was applied to fabricate colloidal submicron particles. Analyses of the fluorescence intensity and thiol group content revealed heat-induced conformational changes of individual protein molecules but not protein aggregation. SEM micrographs confirmed the formation of spherical-shaped submicron and nano-particles. Ultrasound treatment relatively decreased the interfacial tension of prolamin-based particles. Contact angle results suggested the possible superiority of hordein and secalin particles to zein ones in Pickering stabilization. A predominant elastic gel-like behavior (i.e., G'>G″) along with different dependencies on the angular frequency was observed in dynamic frequency sweep test of Pickering emulsions. The shear thinning behavior of all samples was attributed to the deflocculation of oil droplets. The final emulsion characteristics were influenced by the particle type and the applied physical treatments. It was found that hordein-based particles have the ability to develop emulsion-foam (triphasic) structures with the physical stability at least 20 times higher than those prepared by zein-based particles. The results of this study may help in the design of Pickering systems using prolamin-based natural particles.

Enzymatic susceptibility of wheat gluten after subcritical water treatment.

Subcritical water (SCW) hydrolysis is an alternative to traditional methods of protein hydrolysis that uses water as a reaction medium. In this study, the effect of SCW treatment on heat-induced conformational changes in wheat gluten and its relation to enzymatic susceptibility were investigated. The degree of deamidation increased rapidly from 12.5 to 47.4% with increase in the temperature range of 160-220°C. Protein solubility increased in a similar pattern with degree of deamidation and almost all protein was solubilized after treatment with SCW at 200°C. SCW treatment in a particular time-temperature combination results in a significant decrease in enzymatic susceptibility. After SCW treatment at 220°C for 20min, enzymatic susceptibility of gluten protein was exceedingly decreased to nearly complete loss. Because of excess degradation and deamidation and small molecular size (less than 6500Da) many hydrolysis sites disappear and are difficult to access by protease.

S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form – Biochemical and structural studies

Thermotoga maritima is a hyperthermophilic bacterium but its genome encodes a number of archaeal proteins including S-adenosyl-L-homocysteine hydrolase (SAHase), which regulates cellular methylation reactions. The question of proper folding and activity of proteins of extremophilic origin is an intriguing problem. When expressed in E.coli and purified (as a homotetramer) at room temperature, the hyperthermophilic SAHase from T.maritima was inactive. ITC study indicated that the protein undergoes heat-induced conformational changes, and enzymatic activity assays demonstrated that these changes are required to attain enzymatic activity. To explain the mechanism of thermal activation, two crystal structures of the inactive form of T. maritima SAHase (iTmSAHase) were determined for an incomplete binary complex with the reduced cofactor (NADH), and in a mixture of binary complexes with NADH and with adenosine. In contrast to active SAHases, in iTmSAHase only two of the four subunits contain a bound cofactor, predominantly in its non-reactive, reduced state. Moreover, the closed-like conformation of the cofactor-containing subunits precludes substrate delivery to the active site. The two other subunits cannot be involved in the enzymatic reaction either; although they have an open-like conformation, they do not contain the cofactor, whose binding site may be occupied by an adenosine molecule. The results suggest that this enzyme, when expressed in mesophilic cells, is arrested in the activity-incompatible conformation revealed by its crystal structures.

Heat-induced conformational changes of TET peptidase from crenarchaeon Desulfurococcus kamchatkensis.

The effects of heating on the structure and stability of multimeric TET aminopeptidase (APDkam589) were studied by differential scanning calorimetry, tryptophan fluorescence quenching, and dynamic light scattering. Thermally induced structural changes in APDkam589 were found to occur in two phases: local conformational changes, which occur below 70 °C and are not associated with thermal denaturation of the protein, and global structural changes (above 70 °C) induced by irreversible thermal unfolding of the protein accompanied by its spontaneous aggregation. These results may explain the bell-shaped temperature dependence with a maximum at ~70 °C previously observed for enzymatic activity of APDkam589. Interestingly, the thermal unfolding of APDkam589 at about 81.2 °C is accompanied by a so-called blue-shift of about 10 nm-a shift of the Trp fluorescence spectrum toward shorter wavelength. From this point of view, APDkam589 is quite different from most proteins, which are characterized by a long wavelength shift of the spectrum ("red-shift") upon denaturation. The blue-shift of the Trp fluorescence spectrum reflects the changes in the environment of Trp residues, which becomes more hydrophobic upon denaturation. The molecular structure of APDkam589 was determined by X-ray diffraction. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. Therefore, the blue-shift of the Trp fluorescence spectrum can be explained, at least partly, by aggregation of APDkam589, which occurs simultaneously with its thermal denaturation and probably makes the environment of these Trp residues more hydrophobic.

PH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modelling.

In the food industry, glucose oxidase (GOX) is used to improve the shelf life of food materials. The pH- and heat-induced conformational changes of glucose oxidase from Aspergillus niger were quantified by means of fluorescence spectroscopy and molecular dynamics simulations. The phase diagram showed an all-or-none transition process, indicating that pH and temperature largely influence the conformational state of GOX. Shifts in maximum wavelength of Trp, Tyr were registered as the protein encounters a lower pH (pH 4.0), suggesting significant changes of the polarity around the chromophore molecule. Quenching experiments using KI showed higher quenching constants of Trp and flavin adenine dinucleotide upon heating or by changing pH value, and were mainly correlated with the conformational changes upon protein matrix. Finally, valuable insights into the thermal behaviour of GOX were obtained from molecular modelling results. The conformation and structure of GOX protein is dependent upon the pH and heat treatment applied. Molecular dynamics simulation indicated significant changes in the substrate binding region at temperatures over 60 °C that might affect enzyme activity. Moreover, an important alteration of the small pocket hosting the positively charged His(516) residue responsible for oxygen activation appears evident at high temperatures.

Advances in structure–function relationships of tyrosinase from Agaricus bisporus – Investigation on heat-induced conformational changes

A combination of fluorescence spectroscopic measurements, inactivation kinetics and in silico prediction was used in the present study to investigate the heat induced behaviour of tyrosinase from Agaricus bisporus. The phase diagram indicated the existence of at least two distinct species induced by the temperature increase up to 75°C. Regardless of calcium ion presence, the fluorescence intensity results suggest that tyrosinase tends to form aggregates after 10min at 75°C. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of tyrosinase at higher temperature. Detailed insights into tyrosinase structure after performing molecular dynamics simulations, suggest important structural rearrangements of the protein with the temperature increase. The copper coordinating His94 residue was predicted to be involved in salt bridge formation with Glu98, therefore causing significant alteration of the substrate binding site with increasing temperature. These significant changes in tyrosinase structure at temperatures over 60°C might lead to enzyme inactivation.

Effect of Enzymatic Deamidation on the Heat-induced Conformational Changes in Whey Protein Isolate and Its Relation to Gel Properties

The effect of protein-glutaminase (PG) on the heat-induced conformational changes in whey protein isolate (WPI) and its relation to gel properties was investigated. The structural properties of WPI treated with PG were examined by several analytical methods. The analysis of the fluorescence spectrum and the binding capacity of a fluorescent probe demonstrated that deamidation prevented the increase in the fluorescence intensity caused by subsequent heat treatment. Measurements of the molecular weight distribution of WPI showed that PG-treated WPI was not likely to polymerize even after heating. This is thought to be due to an increase in electrostatic repulsion between carboxylic acid groups and a decrease in the formation of disulfide bonds, which results in the decrease in heat-induced aggregation. The properties of heat-induced WPI gels were modified by deamidation. PG-treated WPI gels had a soft texture and a high water-holding capacity in the presence of salts.

Probing thermal behaviour of microbial transglutaminase with fluorescence and in silico methods

Knowledge of transglutaminase behaviour at thermal treatment allows efficient applications in food processing. The heat-induced conformational changes of microbial transglutaminase were studied by fluorescence spectroscopy and a molecular modelling approach. The experimental results indicate the unfolding of transglutaminase in a single-phase reaction, at temperatures over 60 °C. The incidence of conformational changes is also supported by the increase of both intrinsic and 1-anilino-8-naphthalene sulfonate fluorescence intensity with temperature. Changes in the secondary and tertiary structure of transglutaminase were outlined after running molecular dynamics simulations at temperatures ranging from 25 °C to 80 °C. The motif's particularities varied with the temperature, suggesting structural rearrangements of the protein, mainly in helices. The largest deviation from the structure equilibrated at 25 °C was observed at 80 °C.

Fluorescence spectroscopy and molecular modeling investigations on the thermally induced structural changes of bovine β-lactoglobulin

Abstract The heat-induced conformational and structural changes in β-lactoglobulin were analyzed using the fluorescence techniques and the molecular modeling approach. The experimental results confirm a two-state model for heat-induced changes of β-lactoglobulin at pH 6.5. The heat treatment at temperatures higher than 70 °C caused an increase in both intrinsic and ANS fluorescence intensity. The addition of quenching agents was employed to discriminate the fluorescence contributions of the two tryptophan residues of β-lactoglobulin (Trp 19 and Trp 61 ). The addition of acrylamide and KI causes an increase of the quenching constants associated with Trp 19 . This effect is observed at all temperatures studied, but the effect is stronger at temperatures higher than 70 °C. The heat-induced changes in the secondary and tertiary structure were outlined after running molecular dynamics simulations at different temperatures and neutral pH, therefore supporting the experimental observations. Industrial relevance In the food industry, one of the major concerns of whey processors is to produce whey protein products (such as whey protein concentrates or isolates as well as fractions enriched in β-lactoglobulin or α-lactalbumin) with specific functionalities. In this sense, the potential use of β-lactoglobulin as a supplement for special food products has encouraged the study of its physico-chemical and biological properties.

Identification of Mre11 as a Target for Heat Radiosensitization

Thermal radiosensitization is believed to be mediated by an inhibition of double-strand break (DSB) repair, but the exact mechanism of radiosensitization remains to be elucidated. Previously, we demonstrated that proteins of the Mre11/Rad50/Nbs1 complex (MRN) translocate from the nucleus to the cytoplasm in cells have that been heated or heated and then irradiated; this finding led us to propose that heat radiosensitization was due at least in part to translocation of MRN. In the current study, we used leptomycin B to inhibit MRN translocation in heated, irradiated cells, but we found that heat radiosensitization was not altered. Thus enhanced radiosensitivity was not attributed to translocation of MRN proteins. To determine which of the MRN subunits contributed to heat radiosensitization, we compared the extent of heat radiosensitization in wild-type cells with that of cells hypomorphic for Mre11 or Nbs1 or cells in which the level of Rad50 was suppressed. We found that neither Nbs1 nor Rad50 is involved in heat radiosensitization, because a similar amount of heat radiosensitization was observed in cells deficient in those proteins compared to cells expressing normal levels. However, heat radiosensitization was not observed in A-TLD1 cells deficient in Mre11. Measurement of exonuclease activity of purified Mre11 heated at 42.5°C or 45.5°C indicated that the protein is very heat-labile. Immunoprecipitation of Mre11 from heated HeLa cells also revealed that hsp70 associates with Mre11 and that this association is maintained long after heating. Taken together, these findings implicate Mre11 as a target for heat radiosensitization and suggest that heat radiosensitization and inhibition of DSB repair may be mediated by heat-induced conformational changes in Mre11.

Effect of hydrophilic polymer conjugation on heat-induced conformational changes in a protein

End-functional 2-methacryloyloxyethyl phosphorylcholine (MPC) co-polymers containing two different monomer units, 2-hydroxyethyl methacrylate (HEMA) and n-butyl methacrylate (BMA), with varying hydrophilicities were synthesized to investigate the effect of the conjugated hydrophilic polymer on the heat-induced conformational changes of a protein. MPC co-polymer-conjugated proteins containing the HEMA unit (PMH) could withstand thermal conformational changes better than those containing the more hydrophobic BMA unit (PMB). The changes in protein tertiary structures were estimated via the excitation of tryptophan. PMH-conjugated proteins could withstand heat-induced intensity changes better than the PMB-conjugated proteins. Thus, hydrophilic units in the conjugated polymer are probably essential in suppressing the heat-induced conformational changes of a protein. The changes in secondary and tertiary structures of poly(MPC)-(PMPC) and poly(HEMA) (PHEMA)-conjugated proteins were compared to validate the effect of MPC units on heat-induced conformational change. Although the thermally induced conformational changes in the secondary and tertiary structures of PHEMA-conjugated proteins were partially suppressed, the effect on PMPC-conjugated proteins was much greater, with significant conformational preservation. This is due to the specific hydration state of the hydrophilic PMPC chain, which reduces interaction between the protein molecules.

Temperature scanning FTIR analysis of secondary structures of proteins embedded in amorphous sugar matrix

Heat-induced changes in secondary structures of five proteins (bovine serum albumin, BSA; human serum albumin, HSA; myoglobin; ribonuclease A, RNase A; and, β-lactoglobulin, β-Lg) in an amorphous sugar matrix were analyzed by temperature-scanning Fourier transform infrared spectroscopy to elucidate the mechanism of heat-induced conformational change of solid-phase proteins. Three sugars, trehalose, maltose, and dextran (MW 6000), were used. Loss of α-helices due to increasing temperature was observed for BSA, HSA, and myoglobin, which are rich in α-helices. RNase A showed a marked decrease in predominant secondary structural components (β-sheet) with increasing temperature. However, no noticeable changes in the content of secondary structures, except for a slight loss of α-helices, were observed for β-Lg, which is also β-sheet-rich. These heat-induced conformational changes were significant at temperatures above the glass transition temperature. The heat-induced conformational change in BSA dried with sugar appeared time-independent and was clearly different from that due to dehydration and from the thermal conformational change for a solution of BSA. In particular, differences in secondary structural components that increased due to loss of α-helices were noted. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3088–3098, 2009

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.

Structural and Functional Study of Mouse Antidigoxin Monoclonal Antibody Against Thermal Variation

Conformation, heat-induced conformational changes, and the stability of mouse monoclonal antibody against digoxin were analyzed by circular dichroism (CD), fluorescence, UV-Vis spectroscopy, and ELISA techniques. Since both biological function and aggregation depend on the conformational properties of mouse IgG molecules, a series of sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of temperature. CD spectra of IgG show the characteristics expected for beta-proteins. Denaturation experiments performed through optical spectroscopic methods and affinity measurement indicated that the antibody unfolds cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N equilibrium U), where only the native and the unfolded states are significantly populated. Thermal-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. The results show that purified IgG is temperature stable. Of course, affinity assessment as measured by ELISA indicated that increasing the temperature decreases the affinity of IgG toward digoxin.

Effect of sulfoxides on the thermal denaturation of hen lysozyme: A calorimetric and Raman study

A multidisciplinary study of the thermal denaturation of lysozyme in the presence of three sulfoxides with different length in hydrocarbon chain (DMSO, DESO, and DPSO) was carried out by means of DSC, Raman spectroscopy, and SDS–PAGE techniques. In particular, the T d and Δ H values obtained from the calorimetric measurements showed that lysozyme is partially unfolded by sulfoxides but most of the conformation holds native state. The sulfoxide denaturing ability increases in the order DPSO > DESO > DMSO. Moreover, only DMSO and DESO have a real effect in preventing the heat-induced inactivation of the protein and their maximum heat-protective ability is reached when the DMSO and DESO amount is ⩾25% w/w. The sulfoxide ability to act as effective protective agents against the heat-induced inactivation was confirmed by the protein analysis. The enzymatic activity, as well as the SDS–PAGE analysis, suggested that DESO, having a low hydrophobic character and a great ability to stabilise the three-dimensional water structure, is the most heat-protective sulfoxide. An accurate evaluation of the heat-induced conformational changes of the lysozyme structure before and after sulfoxide addition was obtained by the analysis of the Raman spectra. The addition of DMSO or DESO in low concentration resulted to sensitively decrease the heat-induced structural modifications of the protein.