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 Cu2+-(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 fight-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. (1995) Nature 374, 74-77] have shown that these two amino acids are close to each other in the tertiary structure of the related tachykinin NK-1 receptor by engineering a metal ion binding site into the protein. Cys-204 and Cys-276 were found to cross-link in the presence of Cu2+(phenanthroline)(3) oxidant as judged by the fact that the mutant split proteins comigrate with full-length rhodopsin on SDS gels. This method of using Cys mutagenesis and disulfide cross-linking with split proteins to map tertiary interactions should be generally applicable to a number of different membrane proteins.