SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer.
SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride. Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration. Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as a homodimer. As structural criteria, the native dimeric state was assayed by size-exclusion chromatography, chemical cross-linking, tryptophan fluorescence, and circular dichroism. Functional SecA heterodimers were formed of which the individual subunits were tagged with fluorescent dyes to allow measurements of the association state of the monomers by resonance energy transfer using steady-state and time-resolved fluorescence spectroscopy. SecA retained its dimeric structure during translocation, while energy transfer was abolished only by denaturation. The "half-of-the-sites activity" was investigated by constructing heterodimers formed from native and 8-azido-ATP-inactivated SecA. Heterodimers have lost the ability to support translocation of the precursor protein proOmpA in an in vitro translocation system. It is concluded that the dimeric structure is maintained during translocation and required for functionality.
Driessen
A J
AJ
Department of Microbiology, University of Groningen, The Netherlands.
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000648:ultrastructure*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism / Q000648:ultrastructure*; D001692:Biological Transport; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins