F-MAS NMR on proteorhodopsin: enhanced protocol for site-specific labeling for general application to membrane...

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 (19)F-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 (19)F 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 (19)F-MAS NMR.