RESUMO
Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM in bilayers. Separate populations of GpATM peptides were labeled with 2,6-dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor. Quenching of the 2,6-dansyl chloride by the dabsyl group demonstrated an association of the labeled peptides. The quenching was not affected by increases in the amount of lipid present or by unlabeled heterologous peptides but was decreased by the addition of unlabeled GpATM. GpATM was determined to form dimers by fitting the observed energy transfer for a number of donor to acceptor ratios and fitting to the expected number of donor labeled peptides in an oligomer with an acceptor as a function of oligomer number. The finding that the GpATM peptide forms helical dimers in lipid bilayers supports the idea that GpA is a dimer in the erythrocyte membrane. The resonance energy transfer approach may extend to the study of other oligomeric complexes.
Assuntos
Dimiristoilfosfatidilcolina/química , Glicoforinas/química , Bicamadas Lipídicas , Proteínas de Membrana/química , Sequência de Aminoácidos , Biopolímeros , Compostos de Dansil , Transferência de Energia , Indicadores e Reagentes , Dados de Sequência Molecular , p-Dimetilaminoazobenzeno/análogos & derivadosAssuntos
Glicoforinas/química , Proteínas de Membrana/química , Dobramento de Proteína , Sequência de Aminoácidos , Bacteriorodopsinas/química , Análise Mutacional de DNA , Glicoforinas/efeitos dos fármacos , Glicoforinas/genética , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Modelos Químicos , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Relação Estrutura-AtividadeRESUMO
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.
