title: Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.
abstract: Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
authors.forename: M A
authors.initials: MA
authors.lastname: Lemmon
authors.forename: J M
authors.initials: JM
authors.lastname: Flanagan
authors.forename: J F
authors.initials: JF
authors.lastname: Hunt
authors.forename: B D
authors.initials: BD
authors.lastname: Adair
authors.forename: B J
authors.initials: BJ
authors.lastname: Bormann
authors.forename: C E
authors.initials: CE
authors.lastname: Dempsey
authors.forename: D M
authors.initials: DM
authors.lastname: Engelman
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006021:Glycophorins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D006021:Glycophorins; D011993:Recombinant Fusion Proteins; D008836:Micrococcal Nuclease