Quaternary Structure of SecA in Solution and Bound to SecYEG Probed at the Single Molecule Level

SummaryDual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (302 K)Download as PowerPoint slideHighlights► DCFBA allows assessment of the stoichiometry of ligands bound to membrane receptors ► Dimeric SecA binds asymmetrically to the protein-conducting membrane channel SecYEG ► Monomeric SecA binds SecYEG, but dimeric SecA is required for protein translocation ► Protein translocation depends on receptor cycling of the dimeric SecA