Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins.
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The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y.A., Abdulaey, N.G., Feigina, M.Y., Kiselev, A.V. & Lobanov, N.A. (1977) FEBS Lett. 84, 1-4]. Chymotryptic cleavage of bacteriorhodopsin produced two fragments, C-1 (Mr 19,000) and C-2 (Mr 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.
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