Abstract
Missense mutations represent the most common cause of many genetic diseases including cystathionine beta-synthase (CBS) deficiency. Many of these mutations result in misfolded proteins, which lack biological function. The presence of chemical chaperones can sometimes alleviate or even restore protein folding and activity of mutant proteins. We present the purification and characterization of eight CBS mutants expressed in the presence of chemical chaperones such as ethanol, dimethyl sulfoxide, or trimethylamine-N-oxide. Preliminary screening in Escherichia coli crude extracts showed that their presence during protein expression had a significant impact on the amount of recovered CBS protein, formation of tetramers, and catalytic activity. Subsequently, we purified eight CBS mutants to homogeneity (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, and S466L). The tetrameric mutant enzymes fully saturated with heme had the same or higher specific activities than wild type CBS. Thermal stability measurements demonstrated that the purified mutants are equally or more thermostable than wild type CBS. The response to S-adenosyl-L-methionine stimulation or thermal activation varied. The lack of response of R125Q and E176K to both stimuli indicated that their specific conformations were unable to reach the activated state. Increased levels of molecular chaperones in crude extracts, particularly DnaJ, indicated a rather indirect effect of the chemical chaperones on folding of CBS mutants. In conclusion, the chemical chaperones present in the expression medium were able to fully restore the activity of eight CBS mutants by improving their protein folding. This finding could have direct implications for the development of a therapeutical approach to pyridoxine unresponsive homocystinuria.
Highlights
Missense mutations represent the most common cause of many genetic diseases including cystathionine -synthase (CBS) deficiency
Screening of Chemical Chaperones in E. coli Yields Optimal Conditions for Mutant CBS Expression—The impact of the three chemical chaperones at various effective concentrations varied with CBS mutations; in general, 1 and 3% (v/v) ethanol and 3% (v/v) dimethyl sulfoxide (DMSO) showed the best results (Table 1)
The presence of a chemical chaperone in the medium during expression in most cases increased the mutant CBS activity in E. coli crude extracts when compared with its basal activity
Summary
Revealed that a majority of CBS mutants formed large aggregates devoid of heme [12]. These data suggest that the inability of mutants to properly incorporate heme may prevent correct folding, altering the tertiary structure and leading to a loss of biological function, formation of aggregates, and/or degradation. Singh et al [16] showed that osmolytes such as trimethylamine-N-oxide (TMAO), glycerol, or dimethyl sulfoxide (DMSO) increased enzyme activity of I278T and three other CBS mutants by facilitating the formation of active tetramers through stabilization of the folded protein. Mutant CBS Protein Expression and Purification—After the initial chemical chaperone screening, the mutant CBS enzymes were expressed on a large scale (6 liters of the medium) in the presence of the most effective concentration of the selected chemical chaperone and purified as described previously [18]. CBS activities of WT and selected mutants in E. coli crude extracts after expression in the absence or presence of three chemical chaperones. No chaperone 1% ethanol 3% ethanol 1 mM TMAO 10 mM TMAO 100 mM TMAO 1% DMSO 3% DMSO 6% DMSO
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