Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry. Fluorometry can provide unique insights into the structure, assembly and flexibility of complex macromolecules. We continue to develop new techniques and exploit laser-based technology for such studies. 1)This year, we continued our collaborative studies into the Zn++ - dependent activation and oligomerization of HIV- integrase, the enzyme used by the AIDS virus to incorporate itself into human DNA. We employed single-tryptophan containing mutant proteins to make site-specific observations of domain-dependent quenching by a 35bp oligonucleotide from the viral LTR (long terminal repeat) . We found that the core and C-domain sites were quenched by the DNA, but the zinc-finger containing N domain (thought to mediate oligomerization) was not. We prepared several other Trp sites for resonance energy transfer (distance measuring) experiments to help survey the whole complex. 2)We continued collaborative studies into the molten globule states of certain apomyoglobin folding models and protein G, using DAS (decay-associated spectra) and time-resolved anisotropy to look for changes in tryptophan environments and motions. We found that the flexibility of apomyoglobin A helix side chains were restricted not only in native, but also in molten globule states. We found that detailed time-resolved data can resolve and identify the nature of several folding intermediates for GB1 (B1 domain of protein G) that are unseen by other methods. We employed the same techniques to examine the pH-dependent multimerization of R67 DHFR, an enzyme responsible for antibiotic (trimethoprim) resistance. We also began double kinetic studies (fluorescence lifetime and time resolved anisotropy measured during folding reactions) on this protein. 4)We continued our lumisonic imaging project, refined our stopped-flow lifetime and polarization instrument, and developed a new analysis suite (DecayFit). 5) We built and calibrated a new laser-driven upconversion fluorometer ( a sort of laser strobe light for proteins )with subpicosecond resolution. We began measuring very early events in tryptophan fluorescence that reflect on the polarity of its surroundings, to help us better interpret Trp in proteins. - fluorescence protein folding assembly flexibility HIV integrase activation transcription resistance
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