Higher plants, lacking an immune system, have evolved a number of proteins to defend themselves against a range of pathogens including fungi, bacteria and viruses. We propose to solve the X-ray structures of a number of these useful proteins. Together with other methods, including site-directed mutagenesis, we will define the mechanisms of action, facilitating use of these proteins as therapeutic or preventative agents to improve human health. Work will continue on, the structure and action of several ribosome inhibiting toxins, including ricin. Although the structure has been solved and a mechanism of action for the enzymatic A chain (RTA) proposed, additional work is necessary. A number of transition state analogs for the RTA reaction are being synthesized and will be provided for X-ray analysis. The positively charged amino-ribose group will mimic the oxycarbonium ion we have proposed as the transition state intermediate. In addition, 10 base analogs of rRNA substrates will be complexed with RTA for analysis of the extended binding site. Site directed mutations will be made on RTA, expanding on our previous work. A few will probe the substrate binding and catalytic sites, but the new emphasis will be on identifying areas of the protein which mediate membrane translocation. We have developed and tested a translocation assay system using cultured Vero cells. For our analysis, mutants will be made in potential membrane recognition sequences of RTA. Also, mutations will be made to alter the overall protein stability. It is likely that translocation requires partial RTA unfolding and a more stable mutant might be retarded in translocation compared to wild-type. Initial experiments have supported this. Conversion of the surface Leu 161 to Lys results in mutant which has full enzymatic activity but is retarded in the rate of translocation. A range of mutations will be carried out to explore this phenomenon. We have evidence that a true equilibrium denaturation/renaturation protocol can be developed for RTA. This will allow us to measure stability effects for a given mutant. Mutant stability will be correlated with changes in membrane translocation rates as part of our analysis. In addition, correlation of structural differences with folding stability is important in its own right and will contribute to our knowledge of the physical behavior of proteins. We recently solved the X-ray structure of the anti-fungal endochitinase from barley. We propose to refine that structure crystallographically and to form complexes with substrate analogs for analysis. This should help define the mechanism of action of this biomedically useful class of enzymes, about which very little is known. Zeamatin is a pore forming protein isolated from corn; it is the archetypal protein of a newly discovered class of anti-fungal agents. We have obtained useful crystals and plan to solve the crystal structure. Again, this will help us understand the mechanism of membrane insertion and action.
