Time-Resolved Fluorescence Spectroscopy is a powerful tool in biochemistry. Fluorescence emission can provide unique insights into the structure, assembly and flexibility of complex macromolecules. We continue to develop laser-based technology for such studies. This year, our instrumentation was exploited to characterize several important protein (especially DNA-binding) targets. We examined the activity of HIV integrase - the enzyme used by the AIDS virus to paste itself into protected sites in human DNA- by collaborating with two groups. One group developed unique nucleotide analogs with desirable fluorescence that is revealed in interactions with integrase and other DNA-cleaving enzymes; the other group focussed on the Zn2+ dependent assembly of integrase tetramers (and other multimers) thought to be necessary for activity. The latter efforts were supported in our multiparameter stopped-flow lifetime spectrometer, where, for example, we monitored the size of multimers as they fell apart in the seconds after metal ions were rapidly removed by EDTA, a metal sequestering reagent. The same instrument was modified to watch the disassembly process of protein dimers such as tryptophan synthase and tubulin under pressure. We also completed collaborative studies on the contacts made by mutant heat shock factor DNA domains and the transport of prazosin into neurons. We began studies of CMV protease inhibitor-induced structural changes and built a time- resolved phosphorimeter for studies of very large assemblies.
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