Biochemical and biophysical investigations of the SNARE machinery and its interacting proteins

Research interests

The mechanism by which eukaryotic cells transport material between intracellular organelles is of fundamental importance in cell biology. Transport is mediated by vesicles that bud from a donor organelle and afterwards fuse with a target organelle. Currently, it is becoming clear that the underlying molecular machineries involved in the principal aspects of vesicular trafficking are highly conserved among all eukaryotes. Key players during the final step in vesicle trafficking, the fusion of a vesicle with its acceptor membrane, are the so-called SNARE proteins. SNAREs comprise a family of relatively small and mostly membrane-bound proteins. A specialized vesicular transport step occurs at the synapse, where neurotransmitter-loaded synaptic vesicles rapidly release their cargo into the synaptic cleft upon Ca2+-influx. The SNARE proteins involved in this process are syntaxin 1a, synaptobrevin 2, and SNAP-25. Synaptobrevin is a synaptic vesicle protein, whereas syntaxin and SNAP-25 are located in the plasma membrane. The core of the neuronal SNARE complex consists of a very stable four-helix bundle. The parallel orientation of the elongated bundle suggests that SNARE assembly between a vesicle and plasma membrane, starting from the membrane-proximal N-termini, would pull the membranes together (the ‘zipper’ model). So far, however, the evidence for this intuitive mechanism is largely circumstantial. Thus, not surprisingly, the scenario is still controversial.
To come to a better understanding of the molecular events during neuronal exocytosis, we focus on a detailed structural, kinetic, thermodynamic, and phylogenetic characterization of the underlying protein-protein interactions. Ultimately, the kinetic and thermodynamic parameters will allow us to model this complex protein network. In particular, we want to investigate how SNARE assembly takes place, how this process is controlled and catalyzed by other factors and how the tight SNARE complex is disassembled by the ATPase NSF (N-ethylmaleimide Sensitive Factor) and its SNAP-cofactor (Soluble NSF Attachment Protein).
For our studies, we almost exclusively use recombinant proteins expressed in bacteria (E. coli). Next to standard biochemical techniques, we employ spectroscopic (Circular Dichroism and Fluorescence Spectroscopy) and calorimetric (Isothermal Titration Calorimetry) methods. Where feasible, we also make use of high-resolution structural techniques (X-ray crystallography, NMR, cryo EM) in collaborations within the Max-Planck-Institute for Biophysical Chemistry.