Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.
Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.
Resek
J F
JF
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Farahbakhsh
Z T
ZT
Hubbell
W L
WL
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry; D004072:Digitonin; D004578:Electron Spin Resonance Spectroscopy; D005960:Glucosides; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics
D005960:Glucosides; C040358:dodecyl maltoside; D012243:Rhodopsin; D003545:Cysteine; D004072:Digitonin