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.
Highlights
We report here the use of a chimeric protein to show that the presence of just thetransmembrane domain of GpA, fused to a normally monomeric soluble protein, is sufficient to mediate the dimerization of this artificial membrane protein -in SDS
Through measurement of the CD of the chimera we show that the soluble domain is folded into a structure that is notgreatly different from that adopted by staphylococcal nuclease to which no C-terminal fusion has beenmade.We show, through preliminary mutational analysis, that the dimerization is exquisitely sequence specific
We find that themethionine residue previously implicated as important in the dimerization of GpA can be replaced with any nonpolar amino acid with little effectupon the association of the chimera (Table I)
Summary
Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 11, Issue of April 15,pp. 7683-7689,1992 Printed in U.S.A. Each on a different faceof the a-helix disrupt dimerization, of these proteins has a single predicted transmembrane asuggesting a high degree of specificity in the helix- helix, and ion pairs may form between oppositely charged helix interactions. This approachallows the interface residues in the two transmembrane domains. Of Molecdodecyl sulfate; LB, Luria broth;TB, terrific broth; SN, staphylococcal nuclease; PMSF, phenylmethylsulfonyl fluoride; HPLC, highpressure liquid Chromatography; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; EGFR, epidermal growth factor receptor; HER-2, human version of the neu oncogene; BrA, bacteriorhodopsin helix A TM, transmembrane domain; T(is), GpA ular Biophysics and Biochemistry, Yale University, 260 Whitney transmembrane peptide derived from trypsin cleavage; MOPS, 4-. This domain is fused to the C terminus of staphylococcal nuclease via a flexible linkerT.he resulting artificiaml embrane protein
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