A combined computational-biochemical approach offers an accelerated path to membrane protein solubilization

Abstract

AbstractMembrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelinO-acyltransferase (GOAT), a member of the membrane-boundO-acyltransferase (MBOAT) family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank ordered for their proposed ability to solubilize GOAT. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization supporting downstream functional and structural studies.