The long term goal of this proposal is to determine the structural components of the cytochrome P450 2B1 substrate access channel and membrane binding regions and to generate crystals for X-ray structure determination.
The aims are 1) to identify substrate access channel residues in the F helix, F/G loop, and G helix by using site-directed mutagenesis to alter the size of residues and determine the effect on substrate binding rates and metabolism. 2) to determine the membrane binding regions of 2B1 in the N-terminus and F/G loop region using site-directed mutagenesis and to use this information to engineer a soluble, monomeric version of the protein, and 3) to crystallize 2B1 using either traditional crystallization techniques with the modified soluble form of 2B1 or to the lipid cubic phase with the native protein. Detailed knowledge of the relationship between the structure of P450s and their interactions with substrates is required to be able to predict drug interactions and metabolic fate for pharmaceutical purposes. Identifying the structural features that determine substrate access and membrane association is a pivotal remaining question in determining P450 function and predicting substrate metabolism. Elucidation of the structure of cytochrome P450 2B1 will serve as an essential platform for computational modeling of protein-substrate and protein-inhibitor interactions.
