Time Resolved Fluorescence Spectroscopy

Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure, assembly and flexibility of complex macromolecules. 1) This year, we continued our collaborative studies into DNA-protein interactions. Our main target was oligomerization and DNA binding of HIV-integrase, the enzyme used by the AIDS virus to incorporate itself into human DNA. We employed FRET with single-tryptophan mutants to measure site-specific distances between Trp and the end of the viral LTR (long terminal repeat) We found a mixture of two distances for key sites, and we are working to assign them to different functional states of the complex(free, bound and 3'processed). We have also begun using labeled single-cysteine versions of the protein. Our scheme is to build a "scaffold" of distances that the complex fits in, to help us design appropriate drugs. We began studies into the structure and assembly of translin, a DNA-binding protein important in recombination/repair. We learned it is a stable octamer and , using single-Cys mutants, we found the formation of excimers that could only form in a tail-tail assembly. Studies of ssDNA complexes have begun. We also studied local flexibility in the active site of beta-polymerase and protein-induced DNA bending of phased-A tract DNA or normal DNA with factor HU. 2) We completed and published studies of the *femtosecond* dynamics of tryptophan, the most important natural fluorophore in proteins. Using our unique UV upconversion fluorometer, we showed that internal conversion was faster than 100fs, showing that fs measurements of Trp in protein will yield new data about local packing and dielectric environment. We have also made measurements of the 400-femtosecond local librations of platelike molecules (similar to Trp) inside solvent "pockets" to prepare for analogous studies inside proteins. 3) We began collaborative studies into the status of a primary fuel of heart muscle mitochondria- NADH. Our preliminary efforts distinguished free and bound populations of NADH by their different fluorescence lifetimes, and we are moving toward quantifying these reservoirs versus energy state.