A DISTANCE MEASUREMENT BETWEEN SPECIFIC SITES ON THE CYTOPLASMIC SURFACE OF BOVINE RHODOPSIN IN ROD OUTER SEGMENT DISK MEMBRANES

Structural information on mammalian integral membrane proteins is scar ce. As part of work on an alternative approach to the structure of bov ine rhodopsin, a method was devised to obtain an intramolecular distan ce between two specific sites on rhodopsin while in the rod outer segm ent disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodo psin was labeled with a nuclear spin label (P-31) by limited phosphory lation with rhodopsin kinase, Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label . Magic angle spinning P-31-nuclear magnetic resonance revealed the re sonance arising from the phosphorylated protein, The enhancement of th e transverse relaxation of this resonance by the paramagnetic spin lab el was observed. The strength of this perturbation was used to determi ne the through-space distance between the phosphorylation site(s) and the spin label position, A distance of 18+/-3 Angstrom was obtained. ( C) 1997 Elsevier Science B.V.