F-19 NMR-STUDY OF 3-FLUOROPHENYLALANINE-LABELED BACTERIORHODOPSIN

被引:0
作者
ABDULAEVA, GV
SOBOL, AG
ARSENIEV, AS
TSETLIN, VI
BYSTROV, VF
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来源
BIOLOGICHESKIE MEMBRANY | 1991年 / 8卷 / 01期
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Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
F-19 NMR spectra of biosynthetically 3-fluorophenylalanine-labeled bacteriorhodopsin ((F)BR) solubilized in 1 : 1 mixture of chloroform - methanol, 0.1 M LiClO4 were obtained. Signals were assigned in retinyl derivative of (F)BR, Ret-(F)BO, as well as in the fragments obtained both by chemical (NaBH4 and 70% HCOOH/Gu.HCl) and enzymatic (alpha-chymotrypsin, papain and Staphylococcus aureus protease V8) cleavage of polypeptide chain. Isolation of the peptide fragments was achieved using gel permeation chromatography on Sephadex LH-60 in the 1:1 mixture of chloroform - methanol, 0,1 M LiCl. It is found that the cleavage has little or no effect on the chemical shifts of fluorophenylalanine signals, except those of (F)Phe-residues located in the vicinity of cleavage site in the amino acid sequence. This implies that the isolated fragments essentially preserve the conformation of the intact polypeptide chain. The assignment of the fluorine signals for Phe 27,219 and 230 has been performed. The other signals were assigned to the groups of Phe residues: 171 and 208; 88, 135 and 153; 71, 154. 156 and 54 or 42; 42 or 54. Ret-(F)BO derivative spin labeled selectively at Met 163 has been prepared by reacting with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide in absolute ether. The distance (13 +/- 1 angstrom) from spin label to nearest Phe 230 residue was determined based on the analysis of the line broadening of the corresponding signal in the F-19 NMR spectrum.
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页码:30 / 43
页数:14
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