A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.
A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
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McKee
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D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry*; D016254:Mutagenesis, Insertional; D010449:Peptide Mapping / Q000379:methods*; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism; D014162:Transfection
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine