Browsing by Author "Fahlstrom, Carl Ashley"

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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.