Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs.

Abstract

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded β-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.