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.

Membranes protect cells from unwanted loss of genetic material, proteins and nutrients. This creates an individual microclimate inside the cell that enables it to perform specific tasks in the various tissues and organs. However, the desired delimitation of the cell interior also requires that special channels and transporters must be incorporated into the membrane so that the cell can be supplied with nutrients. The installation of these channels and transporters is the responsibility of the so-called translocon. The translocon also has the task of transporting proteins, which are produced in the cell but are needed elsewhere, from the cell interior to the outside. It is not clear how the translocon works, as imaging techniques used up to now rely on frozen samples. The freezing is done in the hope that several thousand translocons remain in the same state, otherwise the signal-to-noise ratio is too bad. Instead of deep-frozen snapshots, averaged over several thousand molecules, we have now been able to create images of a single functional translocon at room temperature. The progress has required us to develop a novel chip in which the translocon floats above an absolutely flat surface in its natural environment, i.e. embedded in a lipid membrane. The summits of other proteins, arranged in a 2-dimensional lattice, serve as pillars which hold the carrier membrane at a defined distance from the base. The images were recorded by means of an atomic force microscope (AFM) that, similar to a turntable, uses a small needle to visualize the translocon. The AFM can scan so fast that even the assembly of the translocon from its two main components in the membrane could be filmed: At first, only the membrane channel which serves for protein transport through the membrane is seen. In a second step, the actual translocation motor approaches the channel and jumps on the channel as if to push a protein through it. However, it leaves its binding site on the channel after a second, and then finally, after several repetitions of the up and down remains in the correct position in anticipation of the substrate to be transported. The developed chip can also be used for many other membrane proteins and thus represents a universal tool that opens up unprecedented possibilities for the research of transport and signaling processes.