Cytochrome P450s are a dynamic family of proteins capable of carrying out different reactions on several classes of substrates, such as the breakdown of xenobiotics and the synthesis of steroids. Many of the human proteins are membrane bound, making them difficult to study in vitro. As an alternative, P450BM3, a bacterial protein, can be used as a model system. By introducing different point mutations, P450BM3 can be made promiscuous and able to catalyze a wider range of reactions than the wild- type (WT) enzyme. Studying the stability, dynamics, and kinetics of these mutations in the presence of different substrates can enhance our understanding of the relationship between substrate choice and stability. In this work, we use two main techniques to investigate the stability of P450BM3 with and without bound substrates: pulse proteolysis and the N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) assay. Pulse proteolysis examines protein degradation of P450BM3 in the presence of chemical denaturants. The results from pulse proteolysis show stabilizing or destabilizing effects of substrates, and allow comparison of the stability of each mutant to the WT P450BM3. The CPM assay monitors thermal denaturation of the protein via a chemical reaction of solvent-exposed cysteines. Together these two assays provide complimentary techniques to assess the stability of P450BM3 as a function of mutation and substrate choice. Our results indicate that wild-type P450BM3 is most stable, but addition of substrates decreases protein stability. In contrast, destabilized, promiscuous mutant forms of P450BM3 exhibit an increase in stability upon addition of substrates. The results of this study suggest that there is trade-off between stability and promiscuity, and suggests that there is an optimal compromise that allows for catalytic function.

Effect of mutations on the stability of P450BM3 as determined by chemical and thermal denaturation

249th National Meeting (2015)

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