Site-resolved ¹³C/¹⁵N solid-state nuclear magnetic resonance analysis of schizorhodopsin 1 in lipid bilayers

Schizorhodopsin (SzR) is a retinal-binding membrane protein that functions as a light-driven inward proton transporter. Solid-state nuclear magnetic resonance (NMR) spectroscopy provides a powerful approach for elucidating atomic-scale structural features of membrane proteins under hydrated lipid bilayer conditions. In this study, we performed a site-specific structural analysis of schizorhodopsin 1 (SzR1) in its resting state based on ¹³C/¹⁵N chemical shift information obtained through extensive sequential resonance assignments. Signal identification was facilitated by a combination of reverse isotopic labeling and dipolar recoupling under magic-angle spinning using REDOR-based experiments, together with conventional three-dimensional solid-state NMR experiments. As a result, resonance assignments were achieved for approximately 80% of the residues. Chemical shift analysis revealed that the carboxyl group of Glu81 in helix C of SzR1 is deprotonated, consistent with its potential role as a proton acceptor, while the guanidinium group of Arg67 is strongly involved in a hydrogen-bonding network in the extracellular half of the protein. These findings demonstrate the capability of solid-state NMR to provide detailed, site-specific information and offer new insights into the structural basis of SzR1 function in a native-like membrane environment.