Abstract
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 coil SecA dinner.
Original language | English |
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Pages (from-to) | 430-439 |
Number of pages | 10 |
Journal | Structure |
Volume | 19 |
Issue number | 3 |
DOIs | |
Publication status | Published - 9-Mar-2011 |
Keywords
- FLUORESCENCE-BURST ANALYSIS
- PROTEIN-TRANSLOCATION CHANNEL
- ESCHERICHIA-COLI
- DIMERIC SECA
- PREPROTEIN TRANSLOCATION
- ATPASE SECA
- CORRELATION SPECTROSCOPY
- COINCIDENCE ANALYSIS
- CRYSTAL-STRUCTURE
- OLIGOMERIC STATE