Fluorescence and Thermodynamics Studies with Proteins

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9407167 Eftink In the last grant period we developed a multi-dimensional spectrophotometer, which enables the simultaneous and automated measurement of multiple types of optical data (circular dichroism, fluorescence, absorbance and light scattering) as a function of temperature, added denaturant, or hydrostatic pressure. It is proposed to further develop this method by adding full spectral fluorescence detection (via an intensified diode array) and by adding the capability of automatic pH (or specific ion) measurement during titration experiments. Additionally we will add a stopped flow accessory to this instrument to enable multi-dimensional monitoring of the time course of the folding/unfolding of macromolecules. %%% It is proposed to extend our studies of the thermodynamics of protein folding by systematically perturbing proteins in ways that are expected to lead to observable populations of folding intermediates, with emphasis on the continued development of new, multi-dimensional spectroscopic methods. We will use these multi-dimensional methods to study the unfolding of selected proteins, which are selected because a) they represent examples of different folding motifs (i.e., alpha helix bundle, beta sheet structure, eight stranded alpha/beta barrel, mixed alpha/beta structures having sub-domains, homodimeric proteins) and because b) they show evidence for a multi-phasic transition under certain conditions. Our intent is to characterize the free energy of coupling between domains/subunits and to appreciate the type of perturbations that affect the interactions between domains/subunits. A second goal is to continue to develop our multi-dimensional spectrophotometer and related methods, to extend our ability to obtain equilibrium and kinetics data for the folding/unfolding of proteins in a multi-dimensional manner. ***