[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.