Structure and Dynamics of Membrane Proteins in Native Cellular Membranes Revealed by <i>In Situ</i> Solid-State NMR

ConspectusMembrane proteins perform essential functions in the complex and heterogeneous environment of the cellular membrane, where their structure and activity are profoundly influenced by the native lipid milieu. Most membrane protein structures in the Protein Data Bank (PDB) have been determined <i>in vitro</i> in membrane-mimetic environments such as detergents. These membrane-mimetic environments can perturb native protein conformations and may result in a loss of function, which in turn leads to misinterpretations of molecular mechanisms. Direct structural investigations of membrane proteins within native cellular membranes are crucial to understanding the undisturbed molecular mechanism. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy provides a powerful platform for <i>in situ</i> studies of membrane proteins. However, achieving high-resolution structure determination in native membranes faces significant obstacles, including limited spectral sensitivity, background interference, and substantial uncertainties in extracting distance restraints from ssNMR spectra. These issues have long hindered complete chemical shift assignments and the determination of high-resolution structures in native environments. To address these issues, our group has established integrated <i>in situ</i> ssNMR methodologies, enabled by the foundational contributions of many research groups. These advances allow for complete resonance assignments, high-resolution structure determination, and residue-specific dynamics analysis of membrane proteins in native cellular membranes. This Account summarizes our contributions over the past decade to <i>in situ</i> ssNMR studies of membrane proteins organized around two complementary themes: methodological advances in <i>in situ</i> ssNMR and the elucidation of how the membrane environment influences molecular mechanisms. The discussion systematically examines (i) recent methodological progress, including selective membrane sample preparation protocols compatible with both ¹³C- and ¹H-detected ssNMR, signal-enhancing pulse sequences, and structural computation methods and (ii) key structural and dynamic insights from <i>in situ</i> ssNMR, including high-resolution structure determination of diverse membrane proteins such as aquaporin Z (AqpZ), the sugar transporter <i>Bj</i>SemiSWEET, and the channel protein MscL, alongside a mechanistic understanding of how the membrane environment regulates their functions. By demonstrating the feasibility of <i>in situ</i> ssNMR for determining membrane protein structures in native settings and providing unprecedented atomic-level insights into their functions, we aim to advance research on membrane-sensitive proteins and promote the broader application of <i>in situ</i> ssNMR methodologies.