A. V. Krivandin

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of α-crystallin became an active field of research over last years. However, fine interactions between α-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between α- and βL-crystallins was studied during thermal denaturation of βL-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of α- and βL-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration ∼14 nm, mean molecular mass ∼4 MDa and maximal size over 40 nm. In pure βL-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the α-crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of βL-crystallin it binds with modified α-crystallin or, alternatively, βL-crystallin complexation and α-crystallin modifications are concurrent. Estimates of the α-βL-crystallin complex size and relative contents of α- and α-βL-crystallins in the complex suggest that several α-crystallin molecules are involved in complex formation.

Heat-induced Complex Formation in Solutions of α- and βL-Crystallins: A Small-Angle X-ray Scattering Study

To gain more insight into molecular mechanisms of chaperone-like activity of α -crystallin, the effect of heating on the protein particle size in solutions of α and β L -crystallins was studied by small-angle X-ray scattering (SAXS). Heating a mixture of α - and β L -crystallins in solution at 60 ° C resulted in the formation of several populations of high-molecular-weight complexes. In contrast, no complex formation was observed at 60 ° C in solutions of β L -crystallin alone. The data obtained provided evidence that α -crystallin at 60 ° C underwent rearrangements that roughly doubled its molecular weight. Based on the results of size estimation for complexes of α - and β L -crystallins, it was concluded that they were assembled via interaction between several macromolecules of α -crystallin. Crystallins are eye lens proteins. α -Crystallin, like heat-shock proteins, functions as a molecular chaperone in preventing aggregation of proteins in solution, induced by heating, UV irradiation, or other denaturing factors [1‐10]. α -Crystallin forms complexes with partially denatured proteins and thereby prevents their aggregation and opacification of the solution. With age, complexes of α -crystallin with other crystallins accumulate in the lens [4]. Chaperone-like activity of α -crystallin is thought to play an important role in maintaining transparency of the eye lens throughout life. Loss of this property by α -crystallin is supposed to contribute to the formation of senile cataract. The study of molecular mechanisms of chaperone-like activity of α -crystallin and especially of the structure of its complexes with denatured proteins is an important physiological problem. Structural information about the complexes can be obtained using SAXS [11]. In this study, we report the results of SAXS analysis of the complexes of α -crystallin with proteins subjected to denaturing treatment.

EPR spectroscopic and X-Ray diffraction studies of carbon fibers with different mechanical properties

The carbon fibers obtained by carbonization of polyacrylonitrile fibers were studied by electron paramagnetic resonance and X-ray diffraction analysis in the range of small and wide scattering angles. Their elastic and strength characteristics were also studied. The concentration of the paramagnetic centers was correlated with the mechanical properties of carbon fibers. The wide-angle X-ray diffraction study did not reveal essential structural differences in the carbon fiber samples with different mechanical properties. At the same time, the small-angle X-ray scattering study showed that the fiber nanostructures with different mechanical properties differ substantially.

A study of melaphene interaction with phospholipid membranes.

218 It is known that presowing treatment of plants with the growth regulator melaphene (melamine salt of bis(oxymethyl)phosphinic acid) at low and ultralow concentrations significantly increases the yield of cereals, legumes, and crucifers, enhancing the stress-resistance of plants under adverse environmental conditions [1, 2]. For substantiated and efficient application of this agent, it is necessary to investigate in detail the mechanism of its action at the molecular and cellular levels. It was shown earlier that the effect of melaphene is determined by the activation of metabolic pathways in stress, which correlated with changes in the microviscosity of annular lipids in cell membranes [3‐5]. The goal of this work was to study the fine structure of model phospholipid membranes under the treatment of melaphene. The range of tested melaphene concentrations was significantly broadened. An additional goal of this study was to reveal possible side effects of melaphene. We studied the interaction of melaphene with the liposomes formed from the individual neutral phospholipid dimyristoylphosphatidylcholine (DMPC) and egg lecithin, which represents a mixture of natural phospholipids. The results of differential microcalorimetric and X-ray diffraction analyses showed that melaphene changes the domain structure of neutral phospholipids in the membrane but has no effect on the next organizational level—the total size of bilayers in multilamellar liposomes formed of natural phospholipids. The effect of melaphene in a broad concentration range (10 ‐21 to 10 ‐3 M) on the conformational rearrangements of the bilayers was assessed by measuring the small-angle X-ray diffraction of egg lecithin liposomes and the thermostability of DMPC liposomes as described in [6‐10]. Small-angle X-ray diffraction analysis of liposomes was performed as described in [8] with some modifications. X-ray diffractograms of dispersed liposomes of phospholipid membranes were obtained using a smallangle X-ray diffractometer equipped with a linear coordinate detector [9]. Diffractograms of dispersed liposomes were normalized by the maximum intensity at the peak of the first diffraction maximum, and the collimation correction for the height of the X-ray beam and detector window was introduced according to [10]. Two diffraction maxima could be distinguished on the liposome dispersion diffractograms of the samples containing melaphene at concentrations 0, 10 ‐21 , 10 ‐18 , 10 ‐12 , and 10 ‐6 M, which represented the first and second orders of reflection of ordered membrane multilayers in liposomes. As seen in Fig. 1, the diffractogram patterns of the studied samples of liposome dispersion coincided, indicating that the membrane structure was the same in all liposome samples. The membrane periodicity interval in liposomes ( D ) was found to be 6.9 nm. The electron density profiles of lipid membranes in the liposomes (Fig. 2) were used to determine the thickness of one membrane, which was calculated as the distance between the electron density peaks corresponding to the position of polar groups in lipids. The membrane thickness in all liposome preparations in the presence of different melaphene concentrations was approximately 4 nm. Thus, the results of small-angle X-ray diffraction analysis showed that melaphene in a broad concentration range ( 10 –21 to 10 –6 M) has no marked effect on the structure of lipid membranes. Differential scanning microcalorimetry of DMPC liposomes was performed by the standard method using a DASM-4 microcalorimeter; the results were subsequently processed using the Microcal Origin 5.0 software [7]. It was found that melaphene at low and ultralow concentrations affects the fine structure of