19F-MAS NMR on Proteorhodopsin: Enhanced Protocol for Site-Specific Labeling for General Application to Membrane Proteins

Proteorhodopsin (PR) is a light-driven proton pump found in near-surface marine gamma-proteobacteria. The green absorbing variant has three cysteines at positions 107, 156 and 175. We probed the accessibility of these residues by F-19-MAS NMR. For this purpose, an efficient but simple protocol for chemical fluorine labeling of accessible cysteines in membrane proteins was established. This one-step reaction was applied to detergent-solubilized PR before reconstitution into phospholipids. All three cysteines could be labeled and showed distinct F-19 chemical shifts with different integral intensities. The accessibility of these cysteines is discussed in the context of a homology model. With the chemical cysteine labeling procedure shown here, an attractive option for site-directed solid-state NMR studies on other membrane proteins is offered due to the high intrinsic sensitivity of F-19-MAS NMR.