Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins

Abstract

NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the β-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here can also serve as a reference for other integral membrane proteins.

Keywords: BamA; Dynamics; Magic angle spinning NMR; Membrane proteins; MsbA; Nuclear magnetic resonance; Protein reconstitution; Solid-state NMR.