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 laser-based technology for such studies.1) This year, we continued our collaborative studies into oligomerization and DNA binding 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 distance between the Trp and a fluorophore at the end of a 35bp oligonucleotide from the viral LTR (long terminal repeat) . We found, unexpectedly, that Trps in either the """"""""DNA binding"""""""" domain or located near the center of the """"""""catalytic core"""""""" domain were not significantly quenched by the Marina-blue labeled DNA, but Trp in the zinc-finger containing N domain (thought to mediate oligomerization) was. Thus the oligomerization domain, not the core, is within about 26A from the DNA end after """"""""3prime processing"""""""", the first step in viral incorporation. These resonance energy transfer (distance measuring) experiments are intended to help us resolve the architecture of the entire DNA-protein complex. This task is impossible with alternative methods. Our current distance constraints will soon be supplanted by similar measurements using labeled single-cysteine versions of the protein (mutants were prepared and recently purified).One can imagine our task as if we were building a """"""""scaffold"""""""" of distances that the complex fits in. Hopefully, this kind of structural insight can help us design appropriate drugs.2) We completed and published collaborative studies into the """"""""molten globule"""""""" states of apomyoglobins 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 (B1domain of protein G) that are unseen by other methods.3) We completed and published similar studies on the pH-dependent multimerization of R67 DHFR, an enzyme responsible for antibiotic (trimethoprim) resistance. We also prepared for """"""""double kinetic"""""""" studies (fluorescence lifetime and time resolved anisotropy measured during folding reactions) on this protein.4) We built and calibrated two new new femtosecond laser-driven """"""""upconversion""""""""fluorometers ( a sort of laser strobe light for Trp fluorescence )with better than 100 femtosecond time resolution. We began measuring very early events (solvent relaxation, internal conversion, picosecond rotations) in tryptophan fluorescence that reflect on the polarity of its surroundings, to help us better interpret the behavior we observe for Trp in proteins. We found picosecond solvent relaxation (color shifts) due to the sudden increase in electric dipole strength of excited Trp. We also found that Trp measurements are a valid reflection of the environment after only a few tens of femtoseconds- so fluorescence will be a valid benchmark when it is used to crosscheck molecular dynamics simulations.
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