A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins

Biochemistry. 1995 Nov 21;34(46):14963-9. doi: 10.1021/bi00046a002.


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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Animals
  • Blotting, Western
  • Cattle
  • Cell Line
  • Cross-Linking Reagents
  • Cysteine / chemistry
  • Disulfides / chemistry
  • Macromolecular Substances
  • Membrane Proteins / chemistry*
  • Mutagenesis, Insertional
  • Peptide Mapping / methods*
  • Rhodopsin / chemistry*
  • Rhodopsin / genetics
  • Rhodopsin / pharmacology
  • Spectrophotometry
  • Transducin / metabolism
  • Transfection


  • Amino Acids
  • Cross-Linking Reagents
  • Disulfides
  • Macromolecular Substances
  • Membrane Proteins
  • Rhodopsin
  • Transducin
  • Cysteine