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Electron transfer (ET) is an important reaction in biological processes such as photosynthesis and respiration. Previous studies have shown that aromatic amino acids, including tryptophan, can serve as hopping sites for long-range ET. The blue copper protein, azurin, is a model protein for the study of tryptophan in a biological ET reaction. Azurin contains a single tryptophan residue (W48) in a hydrophobic pocket that undergoes a photo-induced proton coupled electron transfer (PCET) reaction to generate a long-lived neutral radical. The motivation for this work is to investigate isotopologues in the PCET reaction. A minimal media expression protocol was optimized to produce azurin with perdeuterated tryptophan (W48-d5) in higher yields than previous effort. This was achieved by inclusion of an extended overnight expression at room temperature. Additional isotopologues in which the NH group of the indole ring was exchanged to ND were also generated by following an established protocol. Spectroscopic characterization through UV resonance Raman spectroscopy confirmed that L-Trp-d5 and ND-L-trp-d5 were successfully incorporated as NH-W48-d5, ND-W48-h5, and ND-W48-d5 in azurin. These variants had similar fluorescence quantum yields (within 10%). However, the phosphorescence quantum yields varied, with the W48-d5 variants exhibiting lower yields than W48-h5. Azurin with NH-W48-d5 also displayed decreased quantum yield for ET and the formation of the neutral radical compared to NH-W48-h5. Preliminary data on the ND variants (ND-W48-h5, and ND-W48-d5) were inconclusive because of the inability to reproduce results from the control sample; future experiments will be needed.

Amino acid radicals are important for electron transfer reactions in biological systems. Fundamental processes such as cellular respiration, photosynthesis and magnetoreception of migratory species rely on amino acid radicals to facilitate long distance electron transfer. The tryptophan residue in modified Zn-azurin protein is known to form a stable neutral radical through a photoinduced proton coupled electron transfer reaction in the presence of an exogeneous electron acceptor. A goal of this dissertation is to elucidate the photophysical mechanisms that produces the long-lived neutral radical in Zn-azurin using electronic spectroscopy and to determine the effects of deuteration to the Raman vibrational spectrum and the photoinduced proton coupled electron transfer reaction. The results of a spectroscopic study of the photoinduced proton coupled electron transfer reaction that generates a stable tryptophan neutral radical in Zn-azurin are presented. The quantum yields for electron transfer, tryptophan neutral radical formation, fluorescence, phosphorescence, and the efficiency of proton transfer are reported for Zn-azurin in the presence of the electron acceptors cobalt(III)pentaammine chloride and Cu(II)-azurin. The electron transfer and tryptophan neutral radical quantum yields increased with increasing concentrations of electron acceptor, however, the phosphorescence quantum yield decreased and the fluorescence quantum yield was unaffected. These results indicate that the triplet state is the parent state of electron transfer. A Stern-Volmer analysis of phosphorescence quenching revealed that cobalt(III)pentaammine chloride (KSV = 230,000 M-1) is a more effective quencher than Cu(II)-azurin (KSV = 88,000 M-1). The KSV for Cu(II)-azurin is consistent with reported values for dynamic quenching of tryptophan in azurin, however the KSV for cobalt(III)pentaammine chloride is larger than the reported values for dynamic quenching with smaller charged molecules. Sample

Membrane proteins constitute a significant fraction of cellular proteins due to their vast diversity in function. However, studies of their native structures, folding mechanisms, and dynamics remain a challenge, in part because of their inherent insolubility. One goal of this project is to enhance the tools to study membrane proteins. Small unilamellar vesicles (SUVs) have been used as a bilayer mimetic to investigate membrane protein structures, dynamics, and folding mechanisms. UV resonance Raman spectroscopy (UVRR) has emerged as a powerful technique for probing structures and dynamics of biomolecules. UVRR is particularly valuable for membrane proteins because of the selective enhancement of signal from key aromatic residues and the ability to probe interactions between membrane protein and the lipid. We present practical considerations and guidelines for UVRR data acquisition, including a detailed description of a typical laser setup as well as the process to analyze the tryptophan vibrational modes of a model β-barrel membrane protein, OmpA, unfolded and folded in SUVs. An additional need in studies of membrane protein folding is alternate membrane mimics. Nanodiscs (NDs) are an excellent bilayer alternative to SUVs because of their experimental benefits including homogeneity, optical clarity, low light scattering, and enhanced stability. We combine SDS-PAGE mobility studies with fluorescence, circular dichroism, and UVRR spectroscopy to confirm and characterize the folding of OmpA into NDs. Our studies show similar secondary and tertiary structures in both SUVs and NDs as well as efficient folding yields greater than 88% in both bilayers. The folding of OmpA into NDs was slower compared to in SUVs, and this difference can be attributed to the different bilayer characteristics. Insights into the folding mechanism of OmpA were gained via bimolecular fluorescence quenching with acrylamide quencher. Stern-Volmer analysis utilizing the sphere-of-action model probe

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, allowi

Long-range electron transfer has been observed in several complex biological systems, including photosynthetic complexes to redox enzymes in DNA repair pathways. The efficiency of electron tunneling and hopping through amino acid mediators, including tyrosine and tryptophan, has been reported in the past. In particular, tryptophan residues are redox active and can function as electron transfer (ET) intermediates through progressive conversion to cation and neutral radical. The current work extends upon the published studies of the tryptophan radical at position 48 of the zinc-substituted protein, azurin (denoted ZnIIAz48W) by focusing on the effect of isotopic substitution on electron and protein transfer steps. Two ZnIIAz48W isotopologues of trp-48 are studied here: perdeuterated D5- ZnIIAz48W and singly- deuterated ND- ZnIIAz48W. These isotopologues were characterized by UV- and visible-resonance Raman spectroscopy for both the closed shell and neutral radical forms, respectively. Isotope effects were also observed in the fluorescence and radical quantum yields. The fluorescence and phosphorescence intensities, in the presence and absence of an external electron acceptor, indicate that the triplet state may be involved in the ET and cation radical formation pathways. The quantum yield for formation of D5- ZnIIAz48W is consistent with ET from the triplet state. In the case of ND- ZnIIAz48W, ET also likely occurs from the triplet state, but there is an additional effect because deprotonation of the heavy deuteron reduces the quantum yield for radical formation by a factor of 0.67 relative to NH- ZnIIAz48W. These isotope studies of Trp-48 in azurin help clarify the sequential ET/PT steps for trp-48 in azurin, and provide general insights into this complex biological process.

Long-range electron transfer has been observed in several complex biological systems, including photosynthetic complexes to redox enzymes in DNA repair pathways. The efficiency of electron tunneling and hopping through amino acid mediators, including tyrosine and tryptophan, has been reported in the past. In particular, tryptophan residues are redox active and can function as electron transfer (ET) intermediates through progressive conversion to cation and neutral radical. The current work extends upon the published studies of the tryptophan radical at position 48 of the zinc-substituted protein, azurin (denoted ZnIIAz48W) by focusing on the effect of isotopic substitution on electron and protein transfer steps. Two ZnIIAz48W isotopologues of trp-48 are studied here: perdeuterated D5- ZnIIAz48W and singly- deuterated ND- ZnIIAz48W. These isotopologues were characterized by UV- and visible-resonance Raman spectroscopy for both the closed shell and neutral radical forms, respectively. Isotope effects were also observed in the fluorescence and radical quantum yields. The fluorescence and phosphorescence intensities, in the presence and absence of an external electron acceptor, indicate that the triplet state may be involved in the ET and cation radical formation pathways. The quantum yield for formation of D5- ZnIIAz48W is consistent with ET from the triplet state. In the case of ND- ZnIIAz48W, ET also likely occurs from the triplet state, but there is an additional effect because deprotonation of the heavy deuteron reduces the quantum yield for radical formation by a factor of 0.67 relative to NH- ZnIIAz48W. These isotope studies of Trp-48 in azurin help clarify the sequential ET/PT steps for trp-48 in azurin, and provide general insights into this complex biological process.

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, allowi