Karel Jelínek

Cystathionine β‐synthase mutations: effect of mutation topology on folding and activity

Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine β‐synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS‐soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild‐type enzyme. In contrast to the wild‐type CBS about half of mutants were not activated by S‐adenosylmethionine. Expression at 18°C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency. Hum Mutat 31:1–11, 2010.

Polyelectrolyte shells of copolymer micelles in aqueous solutions: A Monte Carlo study

Multimolecular micelles, formed by polystyrene-block-poly(methacrylic acid) in water, are studied by lattice Monte Carlo method. Electrostatic interactions are calculated in the mean-field approximation by solving the Poisson-Boltzmann equation. The model is parametrized according to available experimental data. The dependence of micellar size on pH and ionic strength is calculated and compared with experimental data. A special attention is devoted to the behavior in solutions with a low ionic strength.

A Monte Carlo study of shells of hydrophobically modified amphiphilic copolymer micelles in polar solvents

Multimolecular micelles in polar solvents formed by polystyrene-block-poly(methacrylic acid) (PS-PMA), hydrophobically modified by a naphthalene tag between blocks and an anthracene tag at the end of PMA block, are studied by a lattice Monte Carlo method. The model is parametrized according to available experimental data and several structural characteristics of the PMA shell together with the fluorometric decay functions are calculated and compared with results obtained for system without anthracene tags. The hydrophobic tags at the ends of shell-forming blocks try to avoid the polar medium and bury in the shell forcing the chains to loop back. The resulting distribution of traps is bimodal with a smaller peak in the vicinity of the core and a larger one farther from the core than the maximum on the distribution of segments.

Monte Carlo study of chain conformations in the swollen middle layer of onion-skin polymeric micelles

Conformations of chains in swollen middle layers of onion-skin micelles were studied by Monte Carlo simulations on a tetrahedral lattice under conditions that mimie real systems of swolten onion-skin micelles. Polymer blocks are modeled as tethered self-avoiding chains, enclosed in a narrow spherical layer. Average density of segments, ca. 0.6, corresponds to swollen micellar systems. Only the excluded volume effect was taken into account since it plays the most important role in dense polymer systems. Individual chains are described by equivalent ellipsoids of gyration. Distributions of the ellipsoid half-axes were calculated during simulations. Results based on a large series of simulations indicate that the middle layer forming blocks may be described as prolonged ellipsoids oriented preferentially perpendicular to the radial direction. Analysis of the data concerning the orientations of end-to-end vectors and distributions of segments within one chain indicates that individual chains are strongly interpenetrated and the multi-chain system is fairly disordered.

Amphiphilic Block Copolymer Micelles with Hydrophobically Modified Shells

The conformational behavior of hydrophobically modified shell-forming poly(methacrylic acid) (PMA) blocks in 1,4-dioxane–water solutions of polystyrene-block-poly(methacrylic acid) (PS-PMA) micelles was studied by a combination of several experimental methods (static and quasielastic light scattering, fluorescence techniques and atomic force microscopy). The nonradiative energy transfer (NRET) studies with fluorescently tagged samples show that the hydrophobic anthracene groups attached to the ends of PMA blocks try to avoid the aqueous medium and return closely to the PS core, forcing PMA chains to loop back. Computer modeling (Monte Carlo and mean-field calculations) was performed to get better insight into the conformational behavior. Computer studies reproduce the behavior on the semi quantitative level.