Effects of polymorphic variation on the thermostability of heterogenous populations of CYP3A4 and CYP2C9 enzymes in solution.

The effect of non-synonymous single nucleotide polymorphisms (SNPs) on cytochrome P450 (CYP450) drug metabolism is currently poorly understood due to the large number of polymorphisms, the diversity of potential substrates and the complexity of CYP450 function. Previously we carried out in silico studies to explore the effect of SNPs on CYP450 function, using in silico calculations to predict the effect of mutations on protein stability. Here we have determined the effect of eight CYP3A4 and seven CYP2C9 SNPs on the thermostability of proteins in solution to test these predictions. Thermostability assays revealed distinct CYP450 sub-populations with only 65-70% of wild-type CYP3A4 and CYP2C9 susceptible to rapid heat-induced P450 to P420 conversion. CYP3A4 mutations G56D, P218R, S222P, I223R, L373F and M445T and CYP2C9 mutations V76M, I359L and I359T were destabilising, increasing the proportion of protein sensitive to the rapid heat-induced P450 to P420 conversion and/or reducing the half-life of this conversion. CYP2C9 Q214L was the only stabilising mutation. These results corresponded well with the in silico protein stability calculations, confirming the value of these predictions and together suggest that the changes in thermostability result from destabilisation/stabilisation of the protein fold, changes in the haem-binding environment or effects on oligomer formation/conformation.