Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry

Site-specific cleavage on the interhelical loop I on the cytoplasmic face o f rhodopsin has been observed after activation of a Cu-phenanthroline tethe red cleavage reagent attached on the cytoplasmic loop IV. The characterizat ion of the reaction products by mass spectrometry, both of the membrane bou nd protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confi rmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-t erminus of rhodopsin. The limited extension of the tether side chain requir es a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 Angstrom. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a sim ulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.