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. email@example.comShow MeSH
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Mentions: Expression of several mutant proteins at 18°C resulted in an increase in their catalytic activity as well as in the amount of tetramers/oligomers; we therefore proposed that these variant proteins may be considered true folding mutations. To evaluate this phenomenon in a more systematic way we have plotted the normalized activity of mutant proteins against the normalized amount of tetramers/oligomers using the combined data from five independent expression experiments. Supp. Figure S2 demonstrates that among the solvent-exposed mutations there are indeed examples of such increase of activities mirroring the increased amounts of tetramers (e.g., p.A114V, p.H65R, p.K102N, p.P78R, p.R125Q, and p.V180A). However, changes in amounts of tetramers of buried mutations were not usually accompanied by corresponding differences in activities. That led us to speculate that the solvent exposure of mutations may be indeed an important determinant of their propensity to misfold and to lose the catalytic activity. We have next performed a regression analysis in which we combined data for all buried and all solvent-exposed mutant proteins, respectively. This analysis has demonstrated that the activity of solvent-exposed mutations correlates quite strongly with the amount of oligomers (r2 = 0.53; see Fig. 3B), and suggests that for this type of mutations enhanced folding achieved by, for example, low temperature or chaperones may result in increased residual activity. In contrast, the mutations buried in the enzyme globule did not exhibit such properties, as there was only a weak correlation between the activity and the amount of oligomers (r2 = .11; see Fig. 3A). The above-described regression analysis suggests that solvent-exposed CBS mutations may be more likely to respond to interventions aimed at correcting their misfolding.
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.org