This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to study the functional coupling between the integral membrane enzyme 2,3-oxidosqualene cyclase (OSC) and its lipid environment. OSC is a key enzyme in the biosynthesis and regulation of cholesterol. It catalyzes the cyclization reaction producing lanosterol, the core skeleton of steroids and hormones. To reach its lipidic substrate, OSC - like all members of the monotopic membrane enzyme family - stably and permanently resides in one leaflet of the bilayer only. Like the other enzymes in this protein family, OSC uses large hydrophobic surfaces to contact the lipid bilayer and utilizes extended hydrophobic channels to shuttle its hydrophobic reactants between its active site and the membrane. The focus of the proposed work is to establish the methodology that will enable the study of the interrelationship between the enzymatic reaction and the properties of the membrane environment. Our long-term goal is to understand the correlation - as mediated by the membrane - between the conformational changes of the protein and the transfer of the substrate and product to and form the lipid bilayer. Initial characterization will focus on expression, purification and reconstitution of the human OSC into membranes of well-defined composition and organization. Using these protocols we will investigate the membrane conditions required for efficient substrate presentation and the effect of the membrane's physicochemical properties on catalysis. To that end 13C-enriched transition state analogues will be utilized which will be enzymatically prepared using OSC mutants that prematurely abort the cyclization reaction. In addition, the compounds binding to OSC and their NMR properties will be characterized in preparation for future solid-state NMR spectroscopy studies. The proposed studies will provide the first quintessential step in characterizing the contribution of the membrane environment to the function of a monotopic membrane enzyme.
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