Reinhard Jahn - US grants

Support is requested for Protein Production. While small-scale preparations of recombinant proteins using standard affinity purifications will be performed in the individual laboratories of the project, all projects require mg-amounts of highly purified recombinant and untagged proteins. The Core will provide technical and scientific support in two ways. First, it will optimize expression of proteins that either do not express at all or only at very low levels, including change of vectors, bacterial strains, co-expression of chaperones, or switching to an eukaryotic expression system. Second, the core will express and purify medium to large scale amounts of proteins using an array of conventional and affinity-based chromatographic methods. The core is equipped with a bacterial fermenter for large-scale expression, two state-of-the-art automated FPLC-systems, and a miniaturaized FPLC (SMART). An array of physico-chemical techniques will be used for initial characterization with respect to folding, oligomerization/aggregation and purity including CD-spectroscopy, multi-angle laser light scattering, and mass spectrometry. It is directd jointly by Drs. Fasshauer and Jahn, who will be assisted by M. Boeddener, an experienced Research Associate with several years of experience in all of the required techniques.

Exocytotic membrane fusion of synaptic vesicles is driven by the SNAREs syntaxin, SNAP-25, and synaptobrevin/VAMP, which assemble in trans between the membranes in a zipper-like fashion, thus pulling the membranes together and overcoming the energy barrier for fusion. In addition, regulatory proteins including synaptotagmin, Munc18, and complexin interact both with the SNAREs and the participating membranes, thus controlling SNARE reactivity and shaping the specific features of cacium-dependent exocytosis of synaptic vesicles. While the zippering model is intuitively satisfying and confirmed by a large body of evidence, the precise sequence of protein-protein interactions and the site of action of the regulatory proteins along the fusion pathway is still only partially understood and controversially discussed. Here we use in-vitro fusion of native secretory vesicles and liposomes reconstituted with purified proteins to isolate partial reactions of the fusion pathway, to understand the structure, dynamics and stoichiometries of the intermediate states of the participating proteins, and to determine the parameters affecting kinetics of each reaction. In the previous funding period, we solved the ci7stal structure of the neuronal SNARE complex with its linkers and transmembrane domains and characterized the interaction of synaptotagmin with SNARE-containing liposomes. Moreover, we developed refined assays for measuring fusion intermediates including fluorescence cross-correlation and cryo electron microscopy, studied the fusion of synaptic vesicles with SNARE-containing liposomes, and interfered with SNARE assembly at the partially or fully zippered state, thus gaining access to intermediate states of the fusion pathway. We now propose to take advantage of these achievements to characterize the intermediate steps in SNARE-mediated fusion and to determine the precise step at which the regulatory proteins synaptotagmin 1, complexin 1, and Munc18-1 operate on membrane fusion catalyzed by neuronal SNAREs. In the first specific aim, we plan to elucidate how SNARE assembly and zippering is connected to membrane docking, hemifusion, and fusion using native and artificial vesicles. In the second specific aim, we will determine the steps in SNARE nucleation and zippering that are acted upon by the regulatory proteins synaptotagmin, MunclS, and complexin. The program will be carried out in close collaboration with Projects 2 and 3 in which complementary approaches are pursued. We expect to obtain essential mechanistic information about the molecular mechanism of neuronal exocytosis that is not obtainable by other approaches..