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Structure. 2011 Mar 9;19(3):430-9. doi: 10.1016/j.str.2010.12.016.

Quaternary structure of SecA in solution and bound to SecYEG probed at the single molecule level.

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Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.


Dual-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.

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