Metrics & MoreArticle Recommendations CONSPECTUS: Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid−protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca 2+ transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by...
