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 scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment 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. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation 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 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.
Albert
A D
AD
Department of Biochemistry, University at Buffalo School of Medicine and Biomedical Sciences, NY, USA.
Watts
A
A
Spooner
P
P
Groebner
G
G
Young
J
J
Yeagle
P L
PL
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine; D004220:Disulfides; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007425:Intracellular Membranes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry; D011725:Pyridines; D012243:Rhodopsin / Q000737:chemistry* / Q000648:ultrastructure; D012374:Rod Cell Outer Segment / Q000737:chemistry* / Q000648:ultrastructure; D013113:Spin Labels
D004220:Disulfides; D005136:Eye Proteins; D008565:Membrane Proteins; D011725:Pyridines; D013113:Spin Labels; C007542:4,4'-dipyridyl disulfide; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D003545:Cysteine