¹⁹ F Ultrafast MAS NMR Reveals the Dynamic Basis of pH-Dependent Regulation in Proteorhodopsin

¹⁹ F NMR spectroscopy is a powerful approach for studying complex biomolecular systems because of its high sensitivity, exceptional responsiveness to local structural changes, and simplified spectra. Here, we demonstrate the application of ¹⁹ F ultrafast MAS NMR to 5-fluorotryptophan-labelled proteorhodopsin reconstituted in lipid bilayers. By assigning 9 of the 10 tryptophan resonances, pH-dependent analyses of chemical shifts, line shapes, and conformational exchange reveal the dynamics of two functionally important residues: W34 in the interprotomer His-Asp-Trp triad and W98 within the retinal-binding pocket. The results identify W34 as a dynamic regulator of proton transport and support a model in which slow ring flipping on the seconds timescale transiently modulates the W34-H75 interaction, thereby acting as a pH-dependent molecular throttle. The spectral characteristics of W98 further suggest that it functions as a dynamic regulator of the photocycle within the retinal-binding pocket. Beyond these mechanistic insights, we show that a MAS rate of 100 kHz markedly enhances the resolution of this ¹⁹ F-labelled membrane protein. Combined with a simple chemical-shift scoring metric and advanced, linear-scaling AF-QM/MM-based ¹⁹ F chemical shift calculations of all sites within this protein, this workflow provides a robust and broadly applicable framework for characterizing membrane protein structure and dynamics in native-like lipid environments.