RESUMO
The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , Citoplasma/metabolismo , Dimerização , Dissulfetos/química , Relação Dose-Resposta a Droga , Cinética , Mutação , Oxigênio/química , Oxigênio/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Dodecilsulfato de Sódio/química , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Ureia/farmacologiaRESUMO
In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.