Hybridizing isotropic and anisotropic solid-state NMR restraints for membrane protein structure determination

Solid-state NMR (ssNMR) techniques for membrane proteins are categorized into two classes: oriented sample (OS) and magic angle spinning (MAS). While these approaches require different sample preparations and pulse sequences, they provide complementary information for the analysis of the structure and topology of membrane proteins embedded in their native lipid bilayer environments. The main difference between the two is that MAS-ssNMR produces highly resolved isotropic spectra by averaging out chemical shift anisotropy (CSA) and dipolar coupling (DC) interactions. In contrast, OS-ssNMR retains this lost information in highly resolved anisotropic spectra by reconstituting membrane proteins into mechanically or magnetically aligned lipid bilayers. Anisotropic parameters from OS-ssNMR report directly on the structural topology of membrane proteins. This feature is especially crucial to describe the membrane architecture and structural transitions of receptors, channels, pumps, and transporters, whose functional dynamics involve topological changes (bend, tilt, rotation, and piston-like motions) of transmembrane domains from inactive to active states. However, OS-ssNMR data are rarely integrated with those from MAS, despite their synergistic nature that would confer a functional relevance to these structures. In this chapter, we illustrate how these techniques can be integrated, providing a mathematical and diagrammatic explanation of anisotropic restraints and a short guide on how to hybridize them with isotropic restraints for routine structure determination.