Botrytis cinerea is a necrotrophic fungus that causes billions of dollars in agricultural damage every year. The BcSnod virulence factors are proteins secreted in the 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.
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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 and membrane proteins. The distinct chemical shifts of phenylalanine, tyrosine, 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 >80% incorporation. Optimization of PSAL phenylalanine expression is underway as well as production of tyrosine and tryptophan. The goal of this project is to make a widely applicable, inexpensive set of reagents to explore macromolecular structure/function/dynamics beyond current capabilities.
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Peptidyl-tRNA is generated from stalled ribosomes. Peptidyl-tRNA hydrolase (Pth) enzymes are essential for the removal of bound peptides and recycling of tRNA. Peptidyl-tRNAs are toxic to cells and without Pth activity, cells die due to impaired translation initiation and slowed protein synthesis due to specific tRNA starvation. A majority of bacteria have one essential Pth enzyme, Pth1, making it a high value drug target. Disrupting Pth1 activity leads to bacterial death and since there is no essential Pth1 homolog found in humans, few side effects are expected from Pth1 inhibitors. We have solved the structure of the Pth1:peptidyl-tRNA complex using small-angle neutron scattering and are continuing with high resolution studies. We are screening natural products (tropical cloudforest and aquatic fungal extracts) expected to contain phytochemicals and secondary metabolites with novel structural motifs and novel mechanisms of bioactivity for Pth1 inhibitors. Numerous extracts have been identified with anti-Pth activity and identification of the active compound is in progress.
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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. 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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Notch Transmembrane Domains
Notch proteins play vital roles in cell signalling. They have been implicated in a number of disease states. Processing of these signalling molecules requires proteolytic cleavage at several sites in or near the membrane. Thus understanding the structure, specificity, and other factors affecting cleavage are key in regulating function.
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