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Spectroscopy of Tryptophan in Electron Transfer and Membrane Protein Folding

Abstract

The tryptophan fluorescence of proteins has been widely used to examine protein structure, ligand binding, and conformational changes. The triplet state is also well suited for examining protein structure and dynamics because of its long lifetime in some proteins, up to seconds. This dissertation focuses on several aspects of the tryptophan triplet, especially the photochemistry and photophysics of this chromophore in electron transfer and membrane protein folding.

Chapter 3 describes the role of the tryptophan triplet state in mediating intermolecular electron transfer (ET). The ET rate across large distances is slow relative to a typical fluorescence lifetime. The photooxidation reaction of tryptophan in mutants of apo- and Zn(II)azurin is shown to involve the triplet state via measurements of triplet absorption and phosphorescence in the presence of an external electron acceptor. The formation of neutral radical is demonstrated to coincide with quenched phosphorescence. The formation kinetics of the triplet state and neutral radical were modeled, and the results of 1×10^7 and 8×10^5 sec-1, respectively, agree with a proposed intermolecular ET pathway (~18 Å) along 10 covalent bonds and two through-space steps.

The tryptophan triplet decay kinetics are known to be different in D2O compared to H2O. This isotope effect is correlated with local solvent accessibility, and can be used to examine changes in hydration during membrane protein folding. Chapter 4 describes experiments on a model tryptophan compound, a membrane-associated peptide (melittin), and a transmembrane protein (OmpA). An isotope effect was present when tryptophan was exposed to bulk solvent, such as in unfolded melittin kH2O/kD2O=0.71, but disappeared when buried in a bilayer, such as in folded melittin kH2O/kD2O=1.07. Additionally, when OmpA was bound to the native molecular chaperone Skp, an isotope effect was absent kH2O/kD2O=1.0 . These results suggest Skp plays a role in desolvating OmpA, allowing OmpA to fold into the bilayer more easily. These data indicate that triplet photophysics may be a general tool to determine changes in hydration for proteins.

Finally, spectroscopy of protein chromophores, including tryptophan, and prosthetic groups reveals local structure and dynamics. Chapter 5 discusses applications of UV and visible resonance Raman spectroscopy to proteins

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