Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity.
Bottom Line: Expression at 18 degrees 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.
Affiliation: First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Institute of Inherited Metabolic Disorders, Prague, Czech Republic. firstname.lastname@example.orgShow MeSH
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Mentions: Using the SDS extraction we observed that all 27 mutant proteins were present in detectable amounts not only in the supernatants but more importantly also in the particulate fractions of the bacterial extracts. The presence of CBS antigen in both fractions is shown in Figure 2; however, this publication gel does not allow inferring either on relative proportions of the mutant proteins in the supernatant and particulate fractions or on their relation to the wild-type enzyme (for details, see the Methods section and legend to Fig. 2). The signal of total SDS-soluble CBS antigen of the mutant proteins was generally somehow decreased with a median of 66% of the wild-type CBS (see Supp. Table S2; gels used for quantification are not shown), although some variant proteins were present in increased quantities. These data suggest that compared to the wild-type enzyme the majority of mutant CBS proteins are less stable in E. coli; however, we cannot exclude that some of them are also less soluble in 3% SDS.
Affiliation: First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Institute of Inherited Metabolic Disorders, Prague, Czech Republic. email@example.com