A new time-resolved fluorescence facility was developed to provide rapid collection and analysis of fluorescence data related to macromolecular size, flexibility, folding and fluctuations. The ability to collect multifrequency phase/modulation data (contemporaneously with our state of the art pulse decay system) was added to our facility, as was the capability to observe fluorescence under 3K bar pressures (to examine volume dependence of folding and subunit-subunit affinities of proteins, along with free volume dependence of lipid fluctuations.) The main time-resolved spectrofluorometer was utilized to study the structure and dynamics of many different proteins, including: gramicidin, a """"""""pore-forming"""""""" peptide; tubulin, a cytoskeletal component whose prefilamentous state can be discerned with bound nile red; arginase and OTCase, enzymes whose linked metabolic feedback is mediated by clear conformational changes identified on our equipment; enzyme I of the phosphotransferase system (work in collaboration with M. Han that led to his being awarded the Lamport Award from the Biophysical Society), a protein whose ligand- dependent subunit association and sulfhydryl reactivity are revealed by nanosecond fluorescence spectra. Protein folding continued to grow as a priority topic in our lab. The metal-stabilized structures of arginase, alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase, and other proteins were perturbed so our rapid-collection instrument could chronicle structural change vs. time. We also continued our inquiry into lipid packing fluctuations, using a unique probe (coronene) that is sensitive to submicrosecond gel fluid equilibration in membranes.
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