Volume 136, number 1 FEBS LETTERS December 1981 A PROBABLE LINKING SEQUENCE BETWEEN TWO TRANSMEMBRANE COMPONENTS OF BACTERIORHODOPSIN Nandini V. KATRE and Robert M. STROUD Department of Biochemistry and Biophysics, University of California, School of Medicine, San Francisco, 94143, USA Received 19 October 1981 1. Introduction Purple membrane from Halobacterium halobium contains predominantly one protein, bacteriorhodop- sin, which acts as a light.driven proton pump [1], and which consists of 7 transmembrane rods of electron scattering density [2]. X-Ray diffraction analysis [3,4] suggests that the rods correspond predominantly to a-helical regions, oriented roughly perpendicular to the membrane plane. However, which parts of the sequence are contained in the rods within the bilayer and which correspond to the linking regions between helices is not known. Enzymatic cleavage [5,6] or chemical modification of the protein, using reagents which preferentially position either into the aqueous, or the lipid phase identify linking or transmembrane regions. We describe specific labelling oftyrosines accessible to lactoperoxidase iodination of bacteriorhodopsin from the aqueous phase. The site of modification is located by separation of peptides using Sephadex LH-60 chromatography and amino acid analysis. 2. Methods 2.1. Isolation of purple membrane Halobacterium halobium strain RI was grown and purple membrane was isolated as in [7]. 2.2. Iodination of tyrosines in purple membrane The tyrosine residues of bacteriorhodopsin were modified using sodium [12sI]iodide and the lactoper- oxidase-glucose oxidase enzyme system [8] immobi- lized on hydrophilic polyacrylamide beads (Enzymo- beads), obtained from BioRad. Purple membrane (2 mg) was suspended in 1 ml 0.2 M phosphate buffer (pH 7.2). The Enzymobead reagent (1 mg) dissolved in 0.5 ml distilled water was added to the membrane suspension. A 10-fold excess of NalZSI over purple membrane was then added and the reaction started by addition of 0.25 ml 2% glucose. After 15 min at room temperature, the enzymobeads were removed by centrifugation 1000 × g for 5 min. The purple membrane was washed several times with phosphate buffer, and concentration assayed using extinction coefficient esT0 = 63 × 10 a cm -1 for the light-adapted form [9]. The same procedure was used to iodinate 20 mg protein, using non-radioactive Nal. 2.3. Identtfication of labeled residues The radioactive and non-radioactive iodinated samples were mixed and initial cleavage of labeled protein was performed by treatment of apomembrane with a-chymotrypsin (Sigma Chemical Co.) at a ratio of 1.5 X 10 -2 mg/mg protein, for 5 h, as described [5 ]. Apomembrane was generated by bleaching in 1 M hydroxylamine hydrochloride, pH 7.0 at 37°C for 2-3 hours [10], and collected by centrifugation at 25 000 × g. The two chymotryptic fragments gener- ated were separated by Sephadex LH-60 chromatog- raphy (column size 2.0 cm diam. × 60 cm length), using 96% formic acid and ethanol (2.5:7) as elution buffer and 2 ml fractions were collected. The larger of the 2 chains (residues 72-248), named (CTI) and the smaller chain (CTII residues 1-71) were separated. CTI was lyophilized, dissolved in 70% HCOOH and treated with cyanogen bromide (CNBr) in 70% HCOOH. A 100-fold molar excess of CNBr over methionine residues was used. The reaction mixture was incubated at 37°C for 24 h, after which it was diluted 10-times with distilled water and lyophilized. Published by Elsevier/North-Holland Biomedical Press 170 00145793/81/0000-0000/$02.75 © 1981 Federation of European BiochemicalSocieties