Botrytis cinerea is a necrotrophic fungs that causes billions of dollars in agricultural damage every year. The BcSnod virulence factors are proteins secreted in teh early stages of infection. Structure/function work on these virulence factors is underway along with characterization of their mechanism of action. Related to the BcSnods is the search for natural product antifungals against B. cinerea. More coming soon.
Pattern Specific Aromatic Labeling
Specific isotope labeling holds many advantages for NMR studies of macromolecular systems. We are developing Pattern Specific Aromatic Labeling (PSAL) to study ever larger systems. The distinct chemical shifts of phenylanine, tryosine, and tryptophan side chains can be implemented as initiators in saturation transfer experiments and are readily distinguished in inter- and intramolecular NOE experiments. Successful production of 1H-13C phenylalanine has been established and recombinant protein expression shows >95% incorporation. Optimization of pattern specifically labeled phenylalanine expression is underway as well as production of tryonsine and tryptophan. The goal of this project is to make a widely applicable, inexpensive set of reagents to explore macromolecular complexes and membrane proteins beyond current circumstances.
Peptidyl-tRNA are generated from stalled ribosomes. Peptidyl-tRNA hydrolase, Pth, is the enzyme essential for the removal of bound peptides from tRNA molecules. Peptidyl-tRNAs are toxic to cells and without Pth "recycling", the cell dies because of impaired translation initiation and slowed protein synthesis due to specific tRNA starvation. The essential activity of Pth makes it a hight value drug target. Disrupting Pth activity leads to bacterial death and since no direct Pth homolog is found in humans, few side effects are expected from inhibitors. In collaboration, we are looking at the high resolution structure of the Pth:peptidyl-tRNA complex using a combination of NMR and small-angle neutron scattering. Also, we are screening tropical cloudforest extracts, expected to contain phytochemicals with novel structural motifs and novel mechanisms of bioactivity, for Pth inhibitors. Numerous extracts have been identified with anti-Pth activity and identification of the active compound is in progress.
The carbohydrate binding protein Scytovirin has potent antiviral activity. The novel fold imparts specificity for MAn4 carbohydrates found on coat glycoprotein of HIV, Ebola, Influenza, and Hepatitis. Biochemical and biophysical studies of the binding and bound complex have lead to structurally engineered mutants that have improved Man4 binding and anti-HIV efficacy. The current research goals are to systematically map mutations of amino acids known to be involved in carbohydrate binding, determine the effects of the number of binding sites on binding and efficacy (multivalency), characterize the structural stability of higher affinity mutants and correlate it to pharmacological activity/longevity, and solve the high-resolution structure of the carbohydrate bound complex.
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