Isotropic Bicelles Stabilize the Functional Form of a Small Multidrug-Resistance Pump for NMR Structural Studies

Abstract

Detergent micelles are commonly used as solubilization agents in biophysical and biochemical studies of membrane proteins, but they do not ideally reproduce the membrane environment and often fail to support the native protein conformation. Bicelles, which are a mixture of short- and long-chain lipids, have long been suggested as a more native-like solubilizing agent for the study of membrane proteins. We tested the use of isotropic bicelles as a system for solution NMR studies of membrane proteins on a small multidrug-resistance protein (Smr), a protein that has so far resisted unambiguous structural characterization by X-ray crystallography. We show that the protein can be reconstituted in its functional form and native oligomerization state in bicelles. With an NMR assignment strategy that makes use of sequential NOE information obtained from a NOESY−TROSY and amino-acid specific information from a TROSY-HNCA experiment, 55% of backbone HN, N, and Cα resonances could be assigned, showing that isotropic...