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Ultrafast Protein Hydration Dynamics Investigated by Femtosecond Fluorescence Spectroscopy
| Title: | Ultrafast Protein Hydration Dynamics Investigated by Femtosecond Fluorescence Spectroscopy |
| Author: | Qiu, Weihong |
| Description: | Protein hydration dynamics is essential for many biological functions. However, understanding protein hydration dynamics has been technically challenged by the time and spatial resolution. This dissertation thus is a systematic investigation of protein hydration dynamics by integrating ultrafast fluorescence spectroscopy with the site-directed mutagenesis method. Using single tryptophan as the local optical probe, we first studied the site-specific solvation dynamics in various different conformations of the prototype peptide melittin and protein human serum albumin; both studies showed a strong correlation between local solvation dynamics and peptide/protein conformation transitions, and the critical role of hydration water in the structural integrity of the peptide/protein. To clarify the molecular interpretation of the solvation dynamics from time-resolved fluorescence studies, we also examined the local solvation dynamics at the surface of protein Staphylococcus nuclease using site-directed mutations; we replaced the local charged residues in close proximity to the tryptophan probe one at a time with an uncharged residue and did not observe significant change in the measured solvation dynamics, thus confirming that solvation correlation functions mainly measure the water dynamics in the protein hydration layer; the solvation dynamics is typically biexponential with the fast time constant resulting from local water relaxation in the hydration layer while the slow time from hydration water network rearrangement tightly coupled with protein fluctuations. To better understand the molecular mechanism of tryptophan fluorescence behaviors in proteins, we then surveyed the tryptophan fluorescence in more than 40 proteins with the femtosecond fluorescence method and molecular dynamics simulations. We were able to identify the carbonyl- and sulfur-containing residues as able quenching groups of tryptophan fluorescence within 100-ps; the former includes glutamine and glutamate residues while the later disulfide bond and cysteine residues. We studied the protein dynamics in human thioredoxin and were able to distinguish four time-overlapping dynamical processes at its active-site and determined their respective time scales; these results elucidated the temporal evolution of hydration water motions, electron transfer reactions and local protein fluctuations at the active site of human thioredoxin, and illustrated continuously synergistic dynamics of biological functions over wide time scales. Building on our understanding of hydration dynamics in isolated proteins or protein mimics, we are applying protein hydration dynamics as a tool to address more biologically relevant questions. Preliminary results are summarized in the final chapter for our efforts to unveil the molecular mechanism of molecular recognition in calmodulin using femtosecond-resolved fluorescence spectroscopy. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1222202233
http://hdl.handle.net/2374.OX/106975 |
| Date: | 2008 |
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