Cytochrome P450s (P450s) in humans are endoplasmic reticulum-bound heme proteins involved in the metabolism of a variety of xenobiotics. Dynamic structural analysis of this protein in aqueous solution is impeded by the aggregation of insoluble protein. By inserting P450s into artificial membranes, a native-like environment is introduced to both simulate cellular conditions and prevent aggregation. Nanodiscs are artificial membranes which have been used previously to solubilize P450s, among a variety of other membrane-associated proteins. They consist of phospholipids, which form a bilayer, the cytochrome P450 protein, and an amphipathic scaffold protein which acts as a belt to stabilize the other components into monodisperse discs. The combined, detergent-solubilized components self-assemble upon removal of the detergent using hydrophobic beads, yielding nanodiscs each containing a single P450 enzyme. Solubilized P450s in nanodiscs will be used in studies on drug substrate binding dynamics in human P450s using infrared spectroscopic techniques. This allows both an environment highly similar to that in the cell, as well as opportunity for the study of interactions between the enzyme, substrate, and membrane using a technique with high spatial and temporal resolution.

Assembly of cytochrome P450s into nanodiscs for infrared spectroscopy

ACS Spring Meeting 2021

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