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 used PCR to engineer seven mutant proteins, each containing only a single tryptophan residue (the native protein contains all seven). After being expressed as """"""""His-tag"""""""" versions, they are being purified as future site-specific structural probes of integrase within DNA complexes. 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 used the same techniques in a new collaboration 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. 3)We continued our collaborative studies of target-induced structure in transcription activation domains (ADs) by examining flexibility of VP16 (from herpesvirus) AD point mutants (alanine replacements). The persistence of the TBP (TATA box binding protein, a target)- induced """"""""freezing"""""""" of these otherwise impaired ADs leads us to believe that target -induced structure is a necessary step- but not sufficient for activation. 4)We continued our """"""""lumisonic"""""""" imaging project, refined our stopped-flow lifetime and polarization instrument, continued our development of """"""""genetic algorithms"""""""" for fluorescence data analysis and began building an ultraviolet """"""""upconversion"""""""" fluorometer ( a sort of laser strobe light for proteins )with potential subpicosecond resolution.
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