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Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity.

Kozich V, Sokolová J, Klatovská V, Krijt J, Janosík M, Jelínek K, Kraus JP - Hum. Mutat. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Institute of Inherited Metabolic Disorders, Prague, Czech Republic. viktor.kozich@lf1.cuni.cz

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Amounts of mutant CBS antigen in fractions of bacterial extracts. A: Mutant proteins expressed at 37°C; B: Results of expression of mutant proteins at 18°C. Top part of panels. The amount of SDS-soluble CBS antigen in particulate (Pel, pellet) and nonparticulate (Sup, supernatant) fractions of bacterial extracts obtained by centrifugation was determined by SDS-PAGE and Western blotting as described in the Methods section. The blots in this figure are only shown to demonstrate the presence of CBS antigen in both fractions and do not reliably reflect the proportion between the fractions; the particulate fractions and the supernatant fractions were loaded on separate gels, whereas in the experimental blots used for quantification these two fractions of each mutant were loaded next to each other on the same gel (data not shown). Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Bottom part of panels. The quaternary structure of CBS mutants was assessed in the water-soluble nonparticulate fraction of bacterial extracts by electrophoresis under native conditions followed by Western blotting; sharply demarcated fractions are tetramers and higher oligomers. Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Solvent exposed mutations, mutations with accessible surface area larger than 40 A2 and/or with relative accessible surface larger than 9% (with the exception of mutations p.C165Y, p.P422L, and p.S466L that belong to buried mutations) buried mutations, remaining mutations from the panel are grouped here (with the exception of mutation p.H65R that belong to solvent exposed mutations). WT, bacterial extracts containing wild-type CBS; pKK 388.1, extracts of bacteria transformed with the empty pKK 388.1 plasmid lacking any CBS.
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fig02: Amounts of mutant CBS antigen in fractions of bacterial extracts. A: Mutant proteins expressed at 37°C; B: Results of expression of mutant proteins at 18°C. Top part of panels. The amount of SDS-soluble CBS antigen in particulate (Pel, pellet) and nonparticulate (Sup, supernatant) fractions of bacterial extracts obtained by centrifugation was determined by SDS-PAGE and Western blotting as described in the Methods section. The blots in this figure are only shown to demonstrate the presence of CBS antigen in both fractions and do not reliably reflect the proportion between the fractions; the particulate fractions and the supernatant fractions were loaded on separate gels, whereas in the experimental blots used for quantification these two fractions of each mutant were loaded next to each other on the same gel (data not shown). Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Bottom part of panels. The quaternary structure of CBS mutants was assessed in the water-soluble nonparticulate fraction of bacterial extracts by electrophoresis under native conditions followed by Western blotting; sharply demarcated fractions are tetramers and higher oligomers. Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Solvent exposed mutations, mutations with accessible surface area larger than 40 A2 and/or with relative accessible surface larger than 9% (with the exception of mutations p.C165Y, p.P422L, and p.S466L that belong to buried mutations) buried mutations, remaining mutations from the panel are grouped here (with the exception of mutation p.H65R that belong to solvent exposed mutations). WT, bacterial extracts containing wild-type CBS; pKK 388.1, extracts of bacteria transformed with the empty pKK 388.1 plasmid lacking any CBS.

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.


Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity.

Kozich V, Sokolová J, Klatovská V, Krijt J, Janosík M, Jelínek K, Kraus JP - Hum. Mutat. (2010)

Amounts of mutant CBS antigen in fractions of bacterial extracts. A: Mutant proteins expressed at 37°C; B: Results of expression of mutant proteins at 18°C. Top part of panels. The amount of SDS-soluble CBS antigen in particulate (Pel, pellet) and nonparticulate (Sup, supernatant) fractions of bacterial extracts obtained by centrifugation was determined by SDS-PAGE and Western blotting as described in the Methods section. The blots in this figure are only shown to demonstrate the presence of CBS antigen in both fractions and do not reliably reflect the proportion between the fractions; the particulate fractions and the supernatant fractions were loaded on separate gels, whereas in the experimental blots used for quantification these two fractions of each mutant were loaded next to each other on the same gel (data not shown). Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Bottom part of panels. The quaternary structure of CBS mutants was assessed in the water-soluble nonparticulate fraction of bacterial extracts by electrophoresis under native conditions followed by Western blotting; sharply demarcated fractions are tetramers and higher oligomers. Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Solvent exposed mutations, mutations with accessible surface area larger than 40 A2 and/or with relative accessible surface larger than 9% (with the exception of mutations p.C165Y, p.P422L, and p.S466L that belong to buried mutations) buried mutations, remaining mutations from the panel are grouped here (with the exception of mutation p.H65R that belong to solvent exposed mutations). WT, bacterial extracts containing wild-type CBS; pKK 388.1, extracts of bacteria transformed with the empty pKK 388.1 plasmid lacking any CBS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2966864&req=5

fig02: Amounts of mutant CBS antigen in fractions of bacterial extracts. A: Mutant proteins expressed at 37°C; B: Results of expression of mutant proteins at 18°C. Top part of panels. The amount of SDS-soluble CBS antigen in particulate (Pel, pellet) and nonparticulate (Sup, supernatant) fractions of bacterial extracts obtained by centrifugation was determined by SDS-PAGE and Western blotting as described in the Methods section. The blots in this figure are only shown to demonstrate the presence of CBS antigen in both fractions and do not reliably reflect the proportion between the fractions; the particulate fractions and the supernatant fractions were loaded on separate gels, whereas in the experimental blots used for quantification these two fractions of each mutant were loaded next to each other on the same gel (data not shown). Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Bottom part of panels. The quaternary structure of CBS mutants was assessed in the water-soluble nonparticulate fraction of bacterial extracts by electrophoresis under native conditions followed by Western blotting; sharply demarcated fractions are tetramers and higher oligomers. Only a single representative sample from two to three independent expression experiments has been loaded for this publication gel. Solvent exposed mutations, mutations with accessible surface area larger than 40 A2 and/or with relative accessible surface larger than 9% (with the exception of mutations p.C165Y, p.P422L, and p.S466L that belong to buried mutations) buried mutations, remaining mutations from the panel are grouped here (with the exception of mutation p.H65R that belong to solvent exposed mutations). WT, bacterial extracts containing wild-type CBS; pKK 388.1, extracts of bacteria transformed with the empty pKK 388.1 plasmid lacking any CBS.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Institute of Inherited Metabolic Disorders, Prague, Czech Republic. viktor.kozich@lf1.cuni.cz

Show MeSH
Related in: MedlinePlus