RDC for Membrane Proteins

Abstract

Rapid progress of lipidic cubic phase crystallography and cryo-electron microscopy in the past decade has drastically lowered the barrier of obtaining high resolution structures of membrane proteins. There are, however, areas of membrane biology that remain largely intractable to these methods. These areas include the transmembrane and juxtamembrane regions of single-pass membrane proteins, small protein domains that dynamically associate with the membrane, as well as intrinsically dynamic membrane proteins such as viroporins and membrane fusogens. For these membrane protein systems, NMR spectroscopy remains the primary biophysical tool for providing structural and dynamic information at residue-specific or even atomic resolution. In theory, NMR studies of membrane proteins are no different from those of soluble proteins, but certain properties specific to membrane proteins present major technical hurdles to structural characterization by NMR. For example, much greater crowding of methyl group resonances, which are a major source of long-range NOEs in conventional NMR-based structure determination, severely limits the amount of assignable tertiary distance restraints. Moreover, the requirement for membrane-mimetic media such as micelles, bicelles, and nanodiscs causes slow molecular tumbling and fast spin coherence relaxation. These properties of membrane proteins, among others, result in much fewer long-range NOE restraints than normally obtainable for soluble proteins. Hence, orientation restraints from residual dipolar couplings (RDCs) are valuable structural constraints that compensate for the sparsity of NOE data. This chapter provides an overview of methods for introducing RDCs for membrane protein samples and how they can be used to complement the distance restraints for structure determination.