A feature of Parkinson’s disease is the presence of fibrillar protein deposits composed mostly of α-synuclein and calcium ions in the brain’s substantia nigra region. Although α-synuclein is natively unfolded, the N-terminal region of the protein is highly helical in the presence of membrane mimics, such as acidic phospholipid vesicles and SDS micelles. The C-terminal region of α-synuclein is known to bind to calcium ions and modulates aggregation. In this thesis, the structure of α-synuclein variants, incorporated with tryptophan and 3-nitrotyrosine as donor and energy acceptor pairs, have been characterized in the presence of SDS micelles, small unilammelar vesicles, and calcium ions by various techniques. Distance distributions extracted from time-resolved fluorescence energy-transfer measurements provide site-specific information on the protein conformations. In addition, similar studies using mutants linked to early onset Parkinson’s disease were also performed to investigate the structural effect caused by these mutations. Furthermore, single tryptophan mutants have been designed as fluorescent reporters. The locations of these different tryptophan residues in the bilayer were probed by lipids labeled with bromine and dinitrophenol quenchers. Finally, preliminary studies of the intramolecular structure of α-synuclein aggregates have been carried out, while elucidation of intermolecular α-synuclein aggregate structures was made possible by the synthesis of new dyes that allow for long-range fluorescent energy transfer.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Gray, Harry B.}, } @phdthesis{10.7907/0E5A-1W62, author = {Shih, Crystal}, title = {Electron Tunneling and Hopping Through Proteins}, school = {California Institute of Technology}, year = {2008}, doi = {10.7907/0E5A-1W62}, url = {https://resolver.caltech.edu/CaltechETD:etd-05122008-105452}, abstract = {Long-range electron tunneling is a central component of processes that are essential for biological function. While many studies have been made to understand electron transfer in proteins, biologically efficient electron transfer at distances exceeding 25 Å remains unobserved in these experiments and hence unresolved. It is proposed that long-range electron transfer is in actuality multistep electron tunneling. What is reported in this thesis is the design, synthesis, and study of many protein systems for the purpose of studying multistep electron tunneling in azurin.
In each system, a histidine has been introduced on the protein for attachment of a high-potential ruthenium or rhenium sensitizer ([Ru(trpy)(tfmbpy)]2+ or [Re(dmp)(CO)3]+); a nitrotyrosine, tryptophan, or tyrosine is placed between the two metal centers on the tunneling pathway. The electron transfer is triggered with the excitation of the metal label with laser light, and the kinetics are monitored, for the most part, by time-resolved UV-VIS spectroscopy.
The first system to empirically demonstrate multistep electron tunneling in proteins was discovered; ultrafast electron transfer is observed between the copper and rhenium centers in the Re124/W122 system; the system was structurally characterized and studied by time-resolved UV-VIS and IR spectroscopies. A two-step tunneling model is proposed; the data sets for the different methods utilized are all in excellent agreement with the model.
Systematic perturbations were made to the working hopping system. It was discovered that nitrotyrosine can participate as an intermediate, but studies to demonstrate its participation in multistep tunneling are not yet fully realized. A second hopping system was discovered in the development of the Re126/W122 system.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Gray, Harry B.}, } @phdthesis{10.7907/deva-c162, author = {Ponce, Adrian}, title = {Electron tunneling in proteins and water}, school = {California Institute of Technology}, year = {2000}, doi = {10.7907/deva-c162}, url = {https://resolver.caltech.edu/CaltechTHESIS:08062014-082923705}, abstract = {
The subject of this thesis is electronic coupling in donor-bridge-acceptor systems. In Chapter 2, ET properties of cyanide-bridged dinuclear ruthenium complexes were investigated. The strong interaction between the mixed-valent ruthenium centers leads to intense metal-to-metal charge transfer bands (MMCT). Hush analysis of the MMCT absorption bands yields the electronic-coupling strength between the metal centers (H_(AB)) and the total reorganization energy (λ). Comparison of ET kinetics to calculated rates shows that classical ET models fail to account for the observed kinetics and nuclear tunneling must be considered.
In Chapter 3, ET rates were measured in four ruthenium-modified highpotential iron-sulfur proteins (HiPIP), which were modified at position His50, His81, His42 and His18, respectively. ET kinetics for the His50 and His81 mutants are a factor of 300 different, while the donor-acceptor separation is nearly identical. PATHWAY calculations corroborate these measurements and highlight the importance of structural detail of the intervening protein matrix.
In Chapter 4, the distance dependence of ET through water bridges was measured. Photoinduced ET measurements in aqueous glasses at 77 K show that water is a poor medium for ET. Luminescence decay and quantum yield data were analyzed in the context of a quenching model that accounts for the exponential distance dependence of ET, the distance distribution of donors and acceptors embedded in the glass and the excluded volumes generated by the finite sizes of the donors and acceptors.
In Chapter 5, the pH-dependent excited state dynamics of ruthenium-modified amino acids were measured. The [Ru(bpy)_(3)] ^(2+) chromophore was linked to amino acids via an amide linkage. Protonation of the amide oxygen effectively quenches the excited state. In addition. time-resolved and steady-state luminescence data reveal that nonradiative rates are very sensitive to the protonation state and the structure of the amino acid moiety.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Gray, Harry B.}, } @phdthesis{10.7907/jh62-an41, author = {Casimiro, Danilo Riguera}, title = {Electron transfer in ruthenium-modified recombinant cytochromes and myoglobins}, school = {California Institute of Technology}, year = {1994}, doi = {10.7907/jh62-an41}, url = {https://resolver.caltech.edu/CaltechTHESIS:06152010-102901683}, abstract = {
The aim of the work described in Chapters 2-4 is to elucidate the role of the protein matrix in determining the distant electronic couplings for intramolecular electron- transfer (ET) reactions. The study focuses on two paradigmatic proteins - myoglobin, a non-ET molecule, and cytochrome c, a mitochondrial electron carrier. Site-directed mutants of human myoglobin were constructed each having a single surface histidine for ruthenium labeling. These histidines are at various distances from the heme center (9.5, His70; 12.7, His48; 15.5 Å, His83). Each mutant was derivatized with a pentaammineruthenium complex and the heme was substituted with a photoexcitable zinc mesoporphyrin. The rates of photoinduced and back ET between the histidine-bound Ru complex and the porphyrin were measured via a laser flash technique. The experimentally derived electronic couplings were found to decay exponentially with the intersite separation.
Three site-directed mutants (Glu66His, Glu66His/Tyr67Phe, and Leu58His) of yeast iso-1-cytochrome c were constructed in order to examine whether intervening aromatic side chains affect the rates of intramolecular ET to a significant extent. The crystal structure of the wild-type protein indicates that the ET pathways involve aromatic side chains - a bridging tryptophan at position 59 (in the case of His58) or a tyrosine at 67 (for His66). Incorporation of the Tyr67Phe mutation in the His66 mutant modifies the ET path in a well defined manner. The rates of intramolecular ET from the ferroheme to ruthenium polypyridine complexes bound to the surface histidines were measured using a laser flash-quench technique. Comparison of the experimentally derived donor-acceptor couplings with those of other previously studied cytochromes does not indicate any significant rate enhancement in the presence of bridging aromatic side chains. Furthermore, the rates correlate reasonably well with the predictions of a σ-tunneling pathway model.
The electronic couplings in these myoglobins and cytochromes were analyzed in the context of current theoretical models. The data on cytochrome c strongly support the presence of specific routes for ET to and from the heme. In contrast, myoglobin provides a homogeneous barrier for ET. A comprehensive analysis of the data on both proteins, however, suggests that the apparent square-barrier nature of the polypeptide matrix of myoglobin is symptomatic of the presence of several competing pathways that effectively cause the electronic couplings to scale with direct intersite separations. These results suggest that more complex factors such as stereoelectronic effects and multiple pathways can contribute significantly to the coupling through a structurally heterogeneous medium such as that of a protein.
In Chapter 5, we have extended the use of a substitution-inert ruthenium polypyridine complex in introducing stabilizing intramolecular crosslinks in a protein. We found a dramatic increase in thermal stability of a yeast iso- 1 -cytochrome c mutant upon crosslinking two adjacent histidines (His39 and His58) on opposite strands of a p- sheet with bis(2,2'-bipyridine)ruthenium complex. The melting point of the Ru-modified cytochrome (72.8 °C) is 23.2 °C higher than that of the unmodified protein. Comparison with another Ru-modified di-histidine mutant suggests that the extent of the effect is largely dependent on the size of the loop generated by the crosslink. The technique should be readily applicable to stabilizing proteins with β-sheets.
Chapter 6 describes a novel strategy for the expression and purification of a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met80—>Ala) in S. cerevisiae. It involves coexpressing in the same plasmid(YEp213) the nonfunctional gene with a functional gene copy for complementation on a nonfermentable carbon medium. The functional gene encodes a product with an engineered metal-chelating site (His39 and His58) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met80—>Ala protein, which possesses a binding site for dioxygen and other exogenous ligands, was produced in quantities sufficient for extensive biophysical characterization. Absorption spectra of several derivatives of this mutant show striking similarities to those of the corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P-450. The new method greatly expands the possible structural changes that can be incorporated into this paradigmatic protein.
Using the technique of time-correlated photon counting, the time-resolved photoluminescence response of n-type gallium arsenide (n-GaAs) in contact with aqueous KOH – Se^2- - Se2^(2-) electrolytes was monitored before and after exposure to aqueous 0.010 M solutions of ruthenium, cobalt and osmium ions. Cobalt ions caused the rate of carrier loss from the GaAs to increase relative to that for untreated samples, as evidenced by faster decays. It was inferred that hole and/or electron transfer to the selenide redox species was catalyzed by the cobalt ions.
A model incorporating ambipolar diffusion, bulk trapping, radiative bimolecular recombination, surface trapping and surface charge transfer and employing a finite-difference algorithm was applied to the photoluminescence decays. Under high intensity illumination, the photoluminescence decays from the gallium arsenide samples with aluminum gallium arsenide overlayers could be fit using the expression for bimolecular kinetics, indicating that radiative recombination dominated the decays in these samples.
For the samples immersed in aqueous KOH – Se^(2-) – Se_2^(2-) solutions, with and without chemisorbed metal ions, a time-dependent increase in the GaAs photoluminescence indicated that changes occurred at the surface during the course of the experiment Prior to metal ion treatment, the lifetime in KOH - Se^(2-) – Se_2^(2-) solutions stabilized at about 3.7 ns, which could be fit with an effective surface hole capture rate constant of 5.1x10^3 cm/sec. After cobalt ion treatment photoluminescence decays with 1/e lifetimes on the order of 0.6 ns were measured and, if it is assumed that surface hole capture dominated the decay, correspond to a surface hole capture rate constant of 1.7x10^5 cm/sec. The 1/e lifetimes for ruthenium and osmium treated surfaces were never observed to be less than 1.5 ns and quickly returned to the 3.7 ns lifetimes observed prior to metal ion treatment It is inferred that the cobalt treated surface is more chemically inert under illumination than either ruthenium or osmium treated surfaces.
Preliminary measurements for GaAs immersed in acetonitrile with and without dimethylferrocene and dimethylferrocenium indicated that the rates of carrier loss at the surface are much higher than they are for the aqueous selenide redox species, even in the absence of the redox species. However, small increases in rate of loss were discernible after addition of redox couple to the acetonitrile, and further work is warranted.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Winkler, Jay Richmond}, }