Our approach to understanding the molecular basis for neurotransmission consists of a combination of structural and biophysical studies. Structural information about the most important complexes between the individual molecular components is first obtained by x-ray crystallography, electron microscopy, or nuclear magnetic resonance spectroscopy. This information provides the framework for investigations targeted at the dynamic aspects of the system, using single-molecule techniques.

Establishment of neural connections is critical for proper brain function, and errors in the process are thought to be associated with autism and other disorders. Nerve impulses are triggered when a presynaptic neuron releases a chemical neurotransmitter into the synapse that is recognized by the postsynaptic neuron. Neurexin and neuroligin, respectively, are presynaptic and postsynaptic connector proteins that extend outside of the cells where they are produced and contact one another to form a physical link across the synapse.

Botulinum neurotoxins (BoNTs) are produced by clostridia and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng / kg, they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. We are studying the mechanism of action of BoNTs, especially receptor recognition and substrate interactions.

We are using single-molecule fluorescence microscopy to observe individual SNARE complexes, associated proteins, and vesicles directly (collaboration with Steven Chu, Lawrence Berkeley National Laboratory). We monitor the binding of the proteins and the fusion of the vesicles to the membrane by illuminating the interaction region with laser light and observing the fluorescent signatures of dyes covalently attached at selected locations on the surface of the SNARE complex. When fluorescent dyes are placed either on the lipids in the vesicle wall or alternatively in the aqueous contents of the vesicle, the spatial distribution of the fluorescent signal indicates the state of mixing of the two lipid bilayers and the degree of containment of the vesicle contents.

Fusion of opposing membranes results in the formation of cis-SNARE complexes that are disassembled for recycling and reactivation by the joint action of SNAP and NSF. NSF is a hexamer and belongs to the AAA (ATPases associated with cellular activities) family of proteins. We are also pursuing structural and functional studies of p97 (also called VCP, valosin-containing protein; VAT in Archaebacteria; and cdc48 in yeast), a distant homolog of NSF. p97 has been implicated in variety of functions, including the extraction of misfolded luminal and membrane proteins from the ER for cytosolic degradation (ERAD).

While SNARE proteins localize specifically throughout the cell, the pairing of these proteins is unlikely to be a major determinant of the specificity of the membrane compartment organization, since SNAREs from different cellular compartments can form complexes in vitro. An initial tethering step has been postulated as the critical determinant in the specificity of membrane fusion. Multimeric protein complexes that are involved in most intracellular trafficking events are essential for this tethering. The tethering complex for exocytosis at the plasma membrane is referred to as the sec6/8 complex, or exocyst, in yeast.

The supply of synaptic vesicles in the presynaptic terminal is limited. With continuous stimulation of the neuron, the available pool would be rapidly depleted. Possible transport of vesicles from the neuronal cell body would be too slow to maintain fast synaptic transmission in the terminal. Instead, the synaptic vesicle membrane that fuses with the plasma membrane is recycled via clathrin-mediated exocytosis.

Small G proteins and their effectors involved in vesicle trafficking.

The release of neurotransmitters triggers a cascade of events in the postsynaptic neuron. The first stage is the binding of the neurotransmitter to a cell surface receptor. The second stage involves an increase of second messengers, the opening or closing of ion channels, or the recruitment of cytoplasmic proteins. The third stage involves the activation of enzymes, typically protein kinases or phosphatases, which mediate the biological response. Thus, the initial interaction of a ligand with a receptor results in amplification of the signal by means of a cascade of responses. These processes are in part regulated by sub-cellular localization and clustering of the cell-surface receptors. Recent studies suggest a major role of protein-binding modules in this process. PSD-95 (PostSynaptic Density-95) is a member of the membrane-associated guanylate kinase (MAGUK) superfamily. In excitatory synapses, PSD-95 clusters glutamate ionotropic receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling molecules and cytoskeletal components.

Structures solved at high resolution and structures solved using highly accurate experimental phases.

Structures of repacking mutants of E. Coli Rop.

Other structures solved in the laboratory.