RESUMO
The twin-arginine translocase (Tat) system is used by many bacteria to translocate folded proteins across the cytoplasmic membrane. The TatA subunit is the predicted pore-forming subunit and has been shown to form a homo-oligomeric complex. Through accessibility experiments using the thiol-reactive reagents 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and Nalpha-(3-maleimidylproprionyl)biocytin toward site-specific cysteine mutants in TatA, we show that the N-terminus of TatA is located in the cytoplasm rather than the previously assumed periplasm. We also confirm previous observations that the C-terminus has a dual topology. By treatment with the membrane uncoupler carbonyl cyanide-m-chlorophenyl hydrazone, we show that the topological state of the C-terminus is dependent on the membrane potential. These results suggest two architectures of TatA in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. Molecular models of both topologies were used to develop and cartoon a homo-oligomeric complex as a channel with a diameter of approximately 50 A and suggest that the double transmembrane helix topology might be the building block for the translocation channel. Additionally, in vivo cross-linking experiments of Gly2Cys and Thr22Cys mutants showed that Gly2, at the beginning of transmembrane helix-1, is in close proximity with Gly2 of a neighboring TatA, as Cys2 cross-linked immediately upon the addition of copper phenanthroline. On the other hand, Cys22, at the other end of the transmembrane helix, took at least 10 min to cross-link, suggesting that a possible movement or reorientation is required to bring this residue into proximity with a neighboring TatA subunit.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Mutação Puntual , Subunidades Proteicas/química , Subunidades Proteicas/genética , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cisteína/química , Cisteína/genética , Citoplasma/química , Citoplasma/metabolismo , Dimerização , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Histidina/química , Potenciais da Membrana , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Desacopladores/farmacologiaRESUMO
Computer simulations are rapidly becoming a standard tool to study the structure and dynamics of lipids and membrane proteins. Increasing computer capacity allows unbiased simulations of lipid and membrane-active peptides. With the increasing number of high-resolution structures of membrane proteins, which also enables homology modelling of more structures, a wide range of membrane proteins can now be simulated over time spans that capture essential biological processes. Longer time scales are accessible by special computational methods. We review recent progress in simulations of membrane proteins.
