Imaging the Translocase Motor SecA at work

An evolutionary conserved heterotrimeric membrane protein complex, called the SecY complex in bacteria, serves to transport secretory and membrane proteins across the plasma membrane. The initial steps of post-translational translocation require binding of the motor protein SecA, which uses ATP hydrolysis to push the nascent chain through the SecY complex. The actual microscopic picture of how that transport occurs has thus far remained enigmatic. The published models of the active quaternary structure of the SecA-SecY complex are either based on high resolution techniques (e.g. X-ray crystallography) which provide static "snapshots" of translocation, or techniques that yield "low resolution" dynamic information of a few amino acid pairs which were either covalently linked or otherwise close enough to allow resonance energy transfer after labelling. We propose to employ a combination of (i) high-speed atomic force microscopy (HS-AFM), (ii) luminescence resonance energy transfer (LRET), and (iii) fluorescence auto- and cross-correlation spectroscopy (FCS) to obtain time-resolved insight into the quaternary state of SecA during translocation. HS-AFM enables visualization of single-protein molecules in liquids at sub-molecular and sub-second temporal resolution permitting us to directly "watch" conformational transitions as they happen in real-time. LRET allows distance measurements between two genetically modified sites of a protein or a protein complex with near Å resolution. The first site accommodates a terbium ion in a lanthanide-binding pocket and the second site is filled with a conventional fluorescent dye. FCS allows precise mobility and concentration measurements, which together with brightness analysis of the diffusing species allows quantification of SecA monomer or dimer binding to the SecY complex. Using these techniques we will directly visualize: I) ligand- and ATP-induced conformational changes of the isolated SecA, II) the oligomeric state of SecA bound to the SecY complex and the relative orientation of the binding partners to each other, and III) conformational changes of SecA during the translocation process of a nascent chain through the channel. The obtained molecular movies, in combination with LRET-assisted docking of available high resolution structures and AFM image simulation techniques will give us a comprehensive picture of ongoing structural dynamics during the translocation process, enabling us to understand the underlying molecular mechanism.

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