Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure, assembly and flexibility of complex macromolecules. This year, 1) We continued collaborative studies into DNA-protein interactions. We published a study of the structure and assembly of translin, a DNA-binding protein important in recombination/repair. We used time-resolved anisotropy to find it is a stable octamer and characterized the reactivity of each of its cysteines. Then, using single-Cys mutants, we found formation of pyrene excimers (nanosecond transient crosslinks) that could only form in a 'tail to tail' quaternary structure. Studies of key distances in translin-ssDNA complexes have also been made via FRET (Forster Resonance Energy Transfer). We studied both the local flexibility of the N helix of beta-polymerase (a segment that controls active site fidelity) and the segmental rotation of its 8kD lyase domain. We found local motions had timescales appropriate for those structural elements, and we found each local motion "freezes" at different times in the catalytic cycle of beta-pol. We related this to the fact that the polymerase also acts as a linear "motor" when processive; publication is pending minor revision. Our main target remains the 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 DNA. A manuscript about this is pending clearance. Recently, we prepared solubility-enhancing mutations for this difficult enzyme, and we used ultracentrifugation to quantify DNA -induced aggregation we had previously seen optically. We have continued preparation of labeled single-cysteine versions for FRET and excimers. Our scheme is to build a "scaffold" of distances that define the complex, to help drug design. 2) We completed studies of the ~400-femtosecond librations of platelike molecules (perylene and tetracene, with sizes similar to tryptophan) inside solvent "pockets" to prepare for similar studies in proteins. Measurement of this libration settles longstanding controversy about anisotropy origins below 0.40 (the "ro defect"). We have begun molecular dynamics simulations with Drs. Brooks and Wu to develop a more appropriate model for the nonexponential libration. We have begun femtosecond upconversion studies of peptides to learn if early electron ejection events (leading to solvated electrons) help explain the "quasistatic self-quenching" we had previously seen in peptides and proteins. 3) We continued collaborative studies with LCE into the status of a primary fuel of isolated heart muscle mitochondria- NADH. Our efforts distinguished free and bound populations of NADH by their different fluorescence lifetimes, and we have quantified these reservoirs during changes in energy state and compartmental concentration.