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abstract
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Fluorescence spectroscopy has made myriad essential contributions to our understanding of molecular and cellular biology. Fundamental properties of intrinsic and extrinsic fluorophores probes in biological systems are exquisitely sensitive to the local bimolecular environment on (sub)-nanometer length scales. Measurement of fluorophore probe properties such as excited state fluorescence lifetime decay, polarization anisotropy, and energy transfer can reflect detailed structural, conformational, motional, binding, and dynamical interactions of macromolecules such as proteins, enzymes, RNA, and DNA. For instance, small changes in the fluorescence lifetime of a probe reveal the characteristics of processes in the local environment at short distances under 10 E (e.g., quenching and dielectric contributions) and long distances up to 100 E (e.g., Forster resonance energy transfer or FRET). This allows direct determination of important parameters of protein-protein binding, complex formation, molecular contact, proximity and overall conformational changes. These lifetime measurements require a highly sensitive lifetime fluorometer with picosecond resolution, such as the requested ISS ChronosBH Fluorescence Lifetime Spectrometer. This automated instrument combines the robust, fast, and proven Time-Correlated Single Photon Counting (TCSPC) method with a novel white light continuously tunable picosecond pulse excitation source that covers the entire visible to near-IR spectrum. The instrument can quickly measure multi-component lifetimes from 40ps to 1us (and longer in phosphorescence mode) with high fidelity from small volume (30uL) and weakly fluorescent samples. Kinetics studies are possible with a stop-flow apparatus. The instrument enables innovative new research as exemplified by 11 different projects from 9 major users. These projects encompass areas as diverse as molecular motions in ion channels, conformational changes in recombinase DNA complexes, amyloid proteins, catalysis regulation of human insulin degrading enzyme, and chemical synthesis of proteins for the development of new fluorescent probes. The need is urgent as there is no instrument of this capability at the University of Chicago or even the greater Chicago area. To increase availability and impact, the instrument will be placed in the interdisciplinary NanoBiology Facility shared core in the Institute for Biophysical Dynamics. Furthermore, it will be integrated in Graduate Biophysics lab curricula, thereby maximizing its exposure and impact on future biological and biomedical researchers.
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