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

19F 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 19F 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 19F-labelled membrane protein. Combined with a simple chemical-shift scoring metric and advanced, linear-scaling AF-QM/MM-based 19F 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. TABLE OF CONTENT GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/739351v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@d1ef03org.highwire.dtl.DTLVardef@a2e5c5org.highwire.dtl.DTLVardef@9b0ecborg.highwire.dtl.DTLVardef@1e837e4_HPS_FORMAT_FIGEXP M_FIG C_FIG