[Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides].

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.

[Changes in physicochemical properties of proteins, caused by modification with alkylhydroxybenzenes].

Kinetic characteristics of model enzymes and physicochemical properties of globular proteins modified by chemical analogues of low-molecular-weight microbial autoregulators (alkylhydroxybenzenes, AHBs) have been studied. C7 and C12 AHB homologues were used, differing in the length of the alkyl radical and the capacity for weak physicochemical interactions. Both homologues affected the degree of protein swelling, viscosity, and the degree of hydrophobicity. The effects depended on the structure of AHBs, their concentration, and pH of the solution, which likely reflects changes in the charge of the protein globule and its solvate cover. Variations of hydrophobicity indices of AHB-modified enzymes (trypsin and lysozyme) were coupled to changes in the catalytic activity. The values of K(M), measured for the enzymes within both AHB complexes, did not change, whereas V(max) increased (in the case of C7 complexes) or decreased (C12 complexes). Possible molecular mechanisms of changes in the physicochemical and catalytic parameters of enzymatically active proteins, induced by modification with structurally distinct AHBs, are described, with emphasis on targeted regulation of functional activity.

[A study of protein structure changes during hydration by means of diffuse X-ray scattering. II. Fourier transform analysis of X-ray scattering data].

Radial distribution functions were deduced by Fourier transform analysis of angular dependences of diffuse x-ray scattering intensities for the following proteins with different hydration degree: water-soluble a-protein myoglobin, water-soluble alpha+beta protein lysozyme, and transmembrane proteins of photosynthetic reaction centers from purple bacteria Rhodobacter sphaeroides and Blastochlorii viridis. The results of Fourier analysis of x-ray scattering intensities give the quantitative characteristics of the mechanisms underlying the influence of water on the formation of biomacromolecules. Water, on the one hand, weakens the intraglobular hydrogen bond net, loosens the protein structure, and increases the internal conformational dynamics. Concurrently water arranges the stability and ordering of the macromolecule. A sharp correlation is observed between the shift of the "first" peak of radial distribution functions, the weakening of the intraglobular hydrogen bond net, the increase in intraglobular mobility, and the appearance of functional activity in macromolecules. The behavior of the "first" peak is similar to that observed in transmembrane protein of reaction center and water-soluble proteins. The "first" peak for transmembrane protein of reaction center reaches its maximum value much faster (at smaller hydration degrees) than for water-soluble proteins. The fast transfer of reaction center protein to its native state during hydration is due to the fact that the dehydrated conformation of reaction center protein is very close to the native one. From a comparison of the radial distribution functions for water, water-soluble proteins and transmembrane proteins, one may conclude that water has the lowest packing density and the lowest order; water-soluble proteins have a larger packing density and are more ordered than water, and transmembrane proteins have the highest degree of packing density and ordering.

[A study of protein structure changes during hydration by diffuse X-ray scattering. I. The intensity and the shape of "10-angstrom" maximum].

The angle dependencies of diffuse x-ray scattering intensities were studied in a wide range of angles from 3 to 80 degrees for water-soluble and membrane proteins with a different structural organization: alpha-helical protein myoglobin, alpha-helical protein serum albumen, alpha + beta protein lysozyme, and transmembrane proteins of photosynthetic reaction centers (RC) from purple bacteria Rhodobacter sphaeroides, and Blastochlorii (Rhodopseudomonas) viridis containing cytocrome c, situated out side the membrane, and for H and L+M subunits of membrane protein of reaction center from Rb. sphaeroides for various hydration degrees. The hydration/dehydration process was studied for water-soluble proteins (within hydration range from h = 0.05 to h = 1). The hydration/dehydration process appears to be reversible. All water-soluble proteins show a 10 angstroms peak, and proteins of reaction center do not show this peak. A quantitative comparable study of the behaviour for of the 10 angstroms peak different proteins the degree of lysozyme hydration increases from h = 0.05 to h = 0.45, the protein structure slightly changes (most probably the motifoffolding), the structure of myoglobin in solution is slightly different from the structure in crystal. By taking into account the changes in the shape and intensity of the 10 angstroms peak only, it is impossible to make the conclusion about structural changes in other proteins studied. A correlation between the structural changes observed and dynamic and functional properties of proteins is discussed.

[Equilibrium fluctuations in myoglobin and lysozyme]. / Ravnovesnye fluktuatsii v mioglobine i lizotsime.

The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins.

[Comparison of dynamic properties of various globular proteins and polyglutamic acid in alpha-helical and coil states. Rayleigh scattering of Mossbauer radiation data]. / Sravnenie dinamicheskikh svoistv razlichnykh globuliarnykh belkov i poliglutaminovoi kisloty v sostoianii alpha-spirali i klubka. Dannye réleevskogo rasseianiia Messbauérovskogo izlucheniia.

Classical model system: Poly-L-glutamic acid (Poly-Glu) was investigated in a disordered coil state (at pH-7.0) and in helix state (at pH 2.0) by Rayleigh scattering of Moessbauer radiation technique. Consider that the coil state of poly-Glu models unfolded (random coil) state and alpha-helix state models the fluctuating secondary structure (during consequent folding of protein) comparative analysis of dynamical properties of poly-Glu in different states with dynamical properties of different proteins in native state (alpha-helical myoglobin and HSA, partially beta-sheet lysozyme) and in intermediate (molten globule) state (alpha-lactalbumin) was performed. This comparison bring some surprising results: native alpha-helical proteins behave itself close to random coil, native partially beta-sheet protein behaves close to fluctuating secondary structure (alpha-helix) and the dynamic behaviour of molten globule state (partially beta-sheet alpha-lactalbumin) is not different from those behaviour of lysozyme and much more rigid than native alpha-helical proteins. As a result one cannot exclude the possibility that folding process and dynamical properties at different steps of the folding are very different for alpha-helical and beta-sheet proteins.

[Study of the dynamics of human serum albumin by coherent Rayleigh dispersion of Mossbauer radiation]. / Izuchenie dinamiki syvorotochnogo al'bumina cheloveka metodom kogerentnogo réleevskogo rasseianiia Messbauérovskogo izlucheniia.

The measurements of angle dependencies of total and elastic Rayleigh scattering of Mossbauer radiation intensities have been performed for human serum albumin (HSA) with hydration degrees h = 0.13 and h = 0.4. The extended model was developed for calculating the inelastic intensity of Rayleigh scattering. Original data for HSA and published data on met-Mb were fitted within the frame of this model. The best agreement with experiment was obtained when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative (mechanical) motions of segments (most probable alpha-helices). Long-range correlated motions are essential at low hydration degree. The possibilities of application of the coherent version of RSMS technique are described.

[Evaluation of the contribution of various types of movement of protein globules into effects observed using the Rayleigh scattering of Mössbauer radiation or the Mössbauer absorption spectroscopy]. / Otsenka vkladov raznykh vidov dvizheniia globul belka v éffekty, nabliudaemye s pomoshch'iu releevskogo rasseianiia messbauérovskogo izlucheniia i messbauérovskoi absorbtsionnoi spektroskopii.

Conditions (regions of hydration degrees and temperatures) are considered at which effects observed in Rayleigh Scattering of Mössbauer Radiation and Mössbauer Absorption Spectroscopy can be attributed to changes in intramolecular mobility, rather than contribution of different types of motions of macromolecules as a whole.

[A study of the effect of solvent composition and viscosity on the molecular dynamics of human serum albumin using Rayleigh scattering of Mössbauer radiation]. / Izuchenie vliianiia sostava i viazkosti rastvoritelia na molekuliarnuiu dinamiku syvorotochnogo al'bumina cheloveka metodom releevskogo rasseianiia messbauérovskogo izlucheniia.

By means of RSMR changes of human serum albumen intramolecular mobility by addition of 1.5% and 7.5% of glutar dialdehyde (GD) in concentrated protein solution, heat denaturation of a protein or substitution of water by water-glycerol solvent with amount of water to glycerol: 1 to 2 were studied. It is shown that the elastic fraction for HSA is changed much less addition of GD or by heat denaturation than by substitution of water solution by water-glycerol. It seems that the observed strong influence of glycerol on intramolecular mobility of HSA is connected mostly with effective dehydration of protein (by substitution of the part of a water solvent by glycerol) and with a small volume decrease of protein (due to preference hydration effect) rather than with the increase of the solvent viscosity.

[Study of protein dynamics using Mössbauer spectroscopy]. / Issledovanie dinamiki belkov metodami messbauérovskoi spektroskopii.

Last experimental results of the study of protein dynamics by Mössbauer absorption spectroscopy and Rayleigh scattering of Mössbauer radiation are reviewed. Dynamical properties of proteins following from the theoretical treatment of these data are described.

[Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of Mössbauer radiation]. / Izuchenie vliianiia gidratatsii na dinamiku nekotorykh globuliarnykh belkov metodom réleevskogo rasseianiia messbauérovskogo izlucheniia.

Hydration relationships of the elastic scattering fraction of Mössbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.

[Role of conformational sub-states on the reaction capacity of protein molecules]. / Rol'konformatsionnykh podsostoianii v reaktsionnoi sposobnosti belkovykh molekul.

A review of the recent data on protein dynamics (mainly myoglobin) by X-ray technique, Mössbauer spectroscopy and Rayleigh scattering of Mössbauer radiation is given. The connection between dynamical and functional properties of biological systems are discussed.

[Debye-Waller factor of Rayleigh scattering of Mössbauer radiation at substances with strong conformation motions]. / O faktore Debaia - Vallera pri réleevskom rasseianii Messbauérovskogo izlucheniia na veshcchestakh, obladaiushchikh sil'noi konformatsionnoi podvizhnost'iu.

A model was developed to describe conformational motion of macromolecules. A new formula was derived for the Debye-Waller factor in accordance with this model. The model proposes the existence of fluidlike motions of fragments within the volume of some A3. The temperature dependence of Rayliegh scattering of Mossbauer radiation was studied and calculated for chromatophores.

[Study of chromophore dynamics by means of Rayleigh scattering of Mossbauer radiation]. / Issledovanie dinamiki khromatoforov s pomoshch'iu réleevskogo rasseianiia messbauérskogo izlucheniia.

The temperature dependence of rayleigh scattering of Mössbauer radiation was studied for two samples of the chromatophores with different relative humidity (P/Ps = 0.35 and 0.94). New type of motion--the transitions between conformational substates was found above 200 degrees K. The model was developed to describe conformational motion in the membranes and membrane proteins. A new formula was derived for the Debye--Waller factor in accordance with this model. The model proposes the existence of fluid-like motions of protein fragments within the volume of some A3, restricted by the profile of the conformational potential. The correlation between decreasing of fR and increasing of electron transition rate was observed.