Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.
Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.
Subczynski
W K
WK
Biophysics Department, Jagiellonian University, Krakow, Poland.
Renk
G E
GE
Crouch
R K
RK
Hyde
J S
JS
Kusumi
A
A
D000818:Animals; D002417:Cattle; D004134:Dimyristoylphosphatidylcholine; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008081:Liposomes; D010100:Oxygen / Q000378:metabolism*; D010713:Phosphatidylcholines; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013113:Spin Labels; D013816:Thermodynamics
D008081:Liposomes; D010713:Phosphatidylcholines; D013113:Spin Labels; D012243:Rhodopsin; C017251:1,2-oleoylphosphatidylcholine; D010100:Oxygen; D004134:Dimyristoylphosphatidylcholine