Theses and Dissertations at Montana State University (MSU)
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Item Computational prediction and experimental measurement of time resolved fluorescence properties of tryptophan and 5-fluoro-tryptophan dipeptides(Montana State University - Bozeman, College of Letters & Science, 2016) Fahlstrom, Carl Ashley; Chairperson, Graduate Committee: Patrik R. Callis; Lee Spangler (co-chair)The widely exploited high sensitivity of the amino acid tryptophan (Trp) fluorescence wavelength and quantum yield on local environment in proteins results in multiexponential decay from two mechanisms: quenching rate heterogeneity and/or spectral relaxation. 5-uorotryptophan (5FTrp) reduces quenching rate heterogeneity by suppression of electron transfer quenching. A comparison of fluorescence properties of Trp and 5FTrp provides a means of differentiating between relaxation and heterogeneity. Four observations concerning the fluorescence of Trp dipeptides required further explanation: decay components of approximately 20 ps, a sub 300 fs 10% loss of quantum yield, the quantum yield for Gly-Trp being significantly lower than Trp-Gly, and the fluorescence wavelength of Trp-X being 10 nm shorter than X-Trp at pH 5. With the goal of distinguishing between electron and proton transfer quenching mechanisms, the time resolved fluorescence--with time resolution of 0.5 ns--for dipeptides was measured in the X-Trp and Trp-X configurations(where X=Leu, Gly, Asp, Arg, Met), with 5FTrp substitution, at pH 5 and pH 10, and in water and D 2O solvent--resulting in 84 distinct species. Molecular dynamics simulations were performed on Gly-Trp and Trp-Gly dipeptides with ground and excited state charges employing multiple force fields. The methods developed by Callis and coworkers were used to calculate the instantaneous rates of electron transfer rates, quantum yield, and fluorescence wavelength. Experimentally three types of multiexponential decay were observed: quenching rate heterogeneity with no relaxation, relaxation only, and combinations thereof. The substitution of 5FTrp for Trp reduced quenching rate heterogeneity, resulting in reduction of short lifetime components, allowing for the observation of relaxation components that were most likely masked in the Trp case. Calculated electron transfer rates support lifetimes of approximately 20 ps, but not those less than 300 fs, and predict a lower quantum yield for Trp-Gly than Gly-Trp. Calculated fluorescence wavelengths reproduce the observed shorter fluorescence wavelength of Trp-X zwitterions. Failure to predict quantum yields may be caused by the inability of the molecular dynamics force fields to reproduce the conformational populations. Results support both relaxation and heterogeneity in Trp dipeptides. 5FTrp is a useful tool in distinguishing between heterogeneity and relaxation.Item Polarized two-photon fluorescence excitation studies of jet-cooled indoles(Montana State University - Bozeman, College of Letters & Science, 1992) Sammeth, David MichaelItem NMR investigations of the role of intrinsic flexibility of the tryptophan repressor(Montana State University - Bozeman, College of Letters & Science, 2012) Goel, Anupam; Chairperson, Graduate Committee: Valerie CopieThe tryptophan repressor protein regulates intracellular concentration of Tryptophan in Escherichia coli by binding to DNA operators and is activated in the presence of high L-Trp concentration by formation of an L-Trp-bound holo-repressor. A Leu to Phe mutation at position 75 generates a temperature-sensitive mutant of TrpR, L75F-TrpR, whereas an Ala to Val mutation only two residue positions further on the protein sequence, at residue position 77, generates a super-repressor mutant of TrpR. Backbone amide dynamics studies on TrpR and the two variants using ¹⁵ N-NMR relaxation techniques at a magnetic field strength of 600 MHz (¹ H Larmor frequency) indicate that all three repressors exhibit comparable diffusion properties, implying that they exhibit very similar global shape, structure, and rotational diffusion properties in both apo- and holo- states, in solution. However, internal backbone amide dynamics of the three apo-repressors reveal small but significant differences in flexibility, which are found primarily for residues spanning the Helix-Turn-Helix DNA-binding domain. These results indicate that the fine-tuning of L-Trp binding interaction is modulated in different ways via small but significant changes in protein flexibility in the two TrpR variants in apo and L-Trp bound forms. Sulfolobus solfataricus, a model organism for Archaea, lives in extreme thermal and acidic environments such as the hot springs of Yellowstone National Park, and is host to diverse archaeal viruses including Sulfolobus spindle shaped virus-1 (SSV1) and Sulfolobus spindle shaped virus-Ragged Hills (SSV-RH). SSV viruses exhibit remarkable morphology and genetic diversity, but are poorly understood as many proteins encoded by their genomes have very little sequence homology to proteins of known functions. We have performed detailed backbone dynamics studies to better understand the mode of ligand recognition by E73, a 73-residue, homodimeric protein encoded within SSV-RH genome. Analysis of backbone dynamics measurements obtained for E73 provides evidence for fast time scale dynamics in the proposed nucleic-acid binding site and motion on the microsecond to millisecond time scale in the loop connecting helices alpha A and alpha B.Item Computation of tryptophan fluorescence quenching by amide and histidine(Montana State University - Bozeman, College of Letters & Science, 2011) Tusell, Jose Ramon; Chairperson, Graduate Committee: Patrik R. CallisTryptophan fluorescence quantum yield is widely used to follow protein folding for the villin headpiece subdomain (HP-35) and a synthetic peptide Ac-W-(A) ₃ -H + -NH ₂ (WH5). These biopolymers have a histidine residue, which is a potent quencher of tryptophan fluorescence, positioned four amino acids away from tryptophan. Experiments assumed that when folding occurs the fluorescence of tryptophan will be quenched by histidine due to the formation of an alpha helix. The reliability of folding and unfolding rate constants determined by tryptophan fluorescence has been called into question by several computational studies. A method to calculate the electron transfer matrix element was developed for different donor/acceptor systems. This method shows that the electron transfer matrix element is sensitive to orientation at close distances and that it does not follow a simple exponential decay with distance. This thesis improved the methods developed by Callis and coworker by conducting 100 ns long simulations for single tryptophan proteins and by modifying the calculation of the fluorescence quantum yield to account for heterogeneity in the calculated electron transfer rates. In addition the method was extended to calculate electron transfer rate constants for histidine quenching by conducting 1 microsecond long simulations of HP-35 and WH5. Calculated tryptophan fluorescence quantum yields for the single tryptophan proteins show better agreement with experiment than was previously reported. Simulations for HP-35 and WH5 indicate that the ability of histidine to quench the fluorescence of tryptophan is surprisingly controlled by the energy gap dependence on the distance that separates them. The energy gap dependence on this distance arises from water solvation around histidine. At large distances this solvation decreases the ability of histidine to accept an electron from tryptophan. Different tryptophan/histidine rotamers control this distance. Even when HP-35 is completely folded much of the time histidine does not quench tryptophan fluorescence contrary to the idea that histidine is only close when HP-35 is folded. The calculated fluorescence quantum yield is sensitive to the distribution of close and far conformations and the rate of exchange between these two conformations. This sensitivity gives credibility to the folding/unfolding rates derived from tryptophan fluorescence quantum yields.