Time filter

Source Type

Kozich V.,Charles University | Sokolova J.,Charles University | Klatovska V.,Charles University | Krijt J.,Charles University | And 3 more authors.
Human Mutation | Year: 2010

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. © 2010 Wiley-Liss, Inc.

Morales-Garcia A.,Czech Institute of Macromolecular Chemistry | Del Corro E.,J. Heyrovsky Institute of Physical Chemistry
Materials Research Express | Year: 2015

Acomputational investigation of β-HP4N7 system is accurately performed using the density functional theory formalism coupled with quantum theory of atoms in molecules. The aim of this work is to understand the behavior of β-HP4N7 under compression. At ambient conditions, theN atoms occupy around 81% of the unit cell volume; for this reason the Natoms are the ones contributing significantly to the bulk properties. The particular tetrahedron (PN4) connection promotes high flexibility to this structure. The atoms composing the solid show significant differences in their compressibilities. The high compressibility (or low resistence to pressure) of theNatoms indicates that the reorganization of these atoms is the key in the densification process that takes place when pressure is increased. When compresing the β-HP4N7 from 0 to 130 GPa the formation of PN6 units is observed, leading to a phase transition. The analysis of the elastic constants reveals the stability of this new phase at 110 GPa. This study shows that β-HP4N7 could be a potential candidate for high pressure synthesis of new phases where P atoms would be 6-fold coordinated. © 2015 IOP Publishing Ltd.

Riesova M.,Czech Institute of Macromolecular Chemistry | Svobodova J.,Czech Institute of Macromolecular Chemistry | Tosner Z.,Charles University | Benes M.,Czech Institute of Macromolecular Chemistry | And 2 more authors.
Analytical Chemistry | Year: 2013

The complexation of buffer constituents with the complexation agent present in the solution can very significantly influence the buffer properties, such as pH, ionic strength, or conductivity. These parameters are often crucial for selection of the separation conditions in capillary electrophoresis or high-pressure liquid chromatography (HPLC) and can significantly affect results of separation, particularly for capillary electrophoresis as shown in Part II of this paper series (Beneš, M.; Riesová, M.; Svobodová, J.; Tesařová, E.; Dubský, P.; Gaš, B. Anal. Chem. 2013, DOI: 10.1021/ac401381d). In this paper, the impact of complexation of buffer constituents with a neutral complexation agent is demonstrated theoretically as well as experimentally for the model buffer system composed of benzoic acid/LiOH or common buffers (e.g., CHES/LiOH, TAPS/LiOH, Tricine/LiOH, MOPS/LiOH, MES/LiOH, and acetic acid/LiOH). Cyclodextrins as common chiral selectors were used as model complexation agents. We were not only able to demonstrate substantial changes of pH but also to predict the general complexation characteristics of selected compounds. Because of the zwitterion character of the common buffer constituents, their charged forms complex stronger with cyclodextrins than the neutral ones do. This was fully proven by NMR measurements. Additionally complexation constants of both forms of selected compounds were determined by NMR and affinity capillary electrophoresis with a very good agreement of obtained values. These data were advantageously used for the theoretical descriptions of variations in pH, depending on the composition and concentration of the buffer. Theoretical predictions were shown to be a useful tool for deriving some general rules and laws for complexing systems. © 2013 American Chemical Society.

Mullerova L.,Czech Institute of Macromolecular Chemistry | Dubsky P.,Czech Institute of Macromolecular Chemistry | Gas B.,Czech Institute of Macromolecular Chemistry
Journal of Chromatography A | Year: 2014

We introduce an easy but highly descriptive model of separation efficiency of dual-selector systems in capillary electrophoresis. The model expresses effective mobilities of analytes in dual-selector mixtures as a function of mixture composition and total concentration. The effective mobility follows the pattern familiar from single-selector systems, while complexation constant and mobility of the complex are replaced by the same but "overall" parameters and a total concentration of the mixture takes the role of a selector concentration. The overall parameters can be either calculated from the individual ones (an arbitrary mixture) or measured directly (a particular mixture). We inspected two model dual-selector systems consisting of heptakis(2,6-di-O-methyl)-β-CD and β-CD and of heptakis(2,6-di-O-methyl)-β-CD and 6-O-α-maltosyl-β-CD, and ibuprofen and flurbiprofen as model analytes (pH 8.2, non-enantioselective separation). Adopting any optimization strategy typically used in single-selector systems and finding an optimal mixture composition and total concentration is perhaps the prime benefit of the model. We demonstrate this approach on the selectivity parameter and show that the model is precise enough to be used in analytical practice. It also results that an electromigration order (reversal) of analytes can exhibit a rather curious dependency on the mixture composition and concentration. Last, the model can be used for better understanding of separation principles in dual-selector systems in general. © 2014 Elsevier B.V.

Mullerova L.,Czech Institute of Macromolecular Chemistry | Dubsky P.,Czech Institute of Macromolecular Chemistry | Gas B.,Czech Institute of Macromolecular Chemistry
Journal of Chromatography A | Year: 2015

Interactions among analyte forms that undergo simultaneous dissociation/protonation and complexation with multiple selectors take the shape of a highly interconnected multi-equilibrium scheme. This makes it difficult to express the effective mobility of the analyte in these systems, which are often encountered in electrophoretical separations, unless a generalized model is introduced. In the first part of this series, we presented the theory of electromigration of a multivalent weakly acidic/basic/amphoteric analyte undergoing complexation with a mixture of an arbitrary number of selectors. In this work we demonstrate the validity of this concept experimentally. The theory leads to three useful perspectives, each of which is closely related to the one originally formulated for simpler systems. If pH, IS and the selector mixture composition are all kept constant, the system is treated as if only a single analyte form interacted with a single selector. If the pH changes at constant IS and mixture composition, the already well-established models of a weakly acidic/basic analyte interacting with a single selector can be employed. Varying the mixture composition at constant IS and pH leads to a situation where virtually a single analyte form interacts with a mixture of selectors. We show how to switch between the three perspectives in practice and confirm that they can be employed interchangeably according to the specific needs by measurements performed in single- and dual-selector systems at a pH where the analyte is fully dissociated, partly dissociated or fully protonated. Weak monoprotic analyte (R-flurbiprofen) and two selectors (native β-cyclodextrin and monovalent positively charged 6-monodeoxy-6-monoamino-β-cyclodextrin) serve as a model system. © 2015 Elsevier B.V.

Discover hidden collaborations