These studies are intended to construct novel """"""""designer"""""""" neurotoxins which can be used for the biochemical and functional characterization of neuronal nicotinic acetylcholine receptors (nAChRs). To elucidate the structure-function relationships of the curaremimetic alpha-neurotoxins, a series of site- directed mutagenesis studies will be performed using a synthetic gene for alpha-bungarotoxin (BGTX) that has been designed to be expressed in E. coli. A strategy of """"""""alanine-scanning"""""""" site-directed mutagenesis will be pursued to reveal those residues in BGTX with clear functional importance for its interaction with muscle-type AChR. In order to fully interpret the effects of these mutations, methods to increase levels of expression and/or refolding will be developed to generate quantities of recombinant BGTX sufficient for efficient structural analysis by NMR and/or X-ray crystallography. New information on the molecular basis for neurotoxin selectivity between muscle and neuronal subtypes of nAChRs will be obtained by constructing """"""""chimeric"""""""" toxins in which selected regions from alpha-bungarotoxin, a toxin which blocks certain subtypes of neuronal nAChRs, will be inserted into a BGTX background. In addition, point mutations will be introduced into BGTX with the view of developing toxins binding with high affinity to neuronal AChRs. Those studies, together with ongoing NMR-based structural analyses of BGTX/receptor peptide complexes, and the observed highly conserved homolgy among muscle and neuronal alpha-subunits, offer the unique opportunity to re-engineer BGTX and to produce novel toxins selective for one or more of the neuronal AChRs.
