Use of tryptophan phosphorescence to assess protein-solvent dynamic coupling in native and mutant glucokinase
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Lewerissa, Karina Bianca.
Use of tryptophan phosphorescence to assess protein-solvent dynamic coupling in native and mutant glucokinase. Retrieved from
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TitleUse of tryptophan phosphorescence to assess protein-solvent dynamic coupling in native and mutant glucokinase
Date Created2022
Other Date2022-01 (degree)
Extent188 pages : illustrations
DescriptionTryptophan phosphorescence decay rates had been used to observe protein dynamics inglucokinase (GK) enzyme, a multi tryptophan protein with 3 tryptophan residues at sequence positions 99, 167, and 257. Three GK mutant enzymes, namely W99, W167, and W257, that contain only one tryptophan residue were used to investigate how local environment at a specific site in the protein determines the spectral properties of tryptophan. The intensity decay rates of tryptophan phosphorescence in those enzymes in different conformational states (free, bound to glucose, and denatured by heat) were collected over a wide range of temperature from 77K, from glassy state through rubbery state far above the solvent glass transition (Tg) of the cryosolvent. Decay rates were interpreted in terms of dynamic quenching by local matrix molecular mobility.
Data show that the molecular mobility of the enzymes as a function of temperature can be separated into 3 kinetic regimes, which we name as process I, II, and III. The dynamic behaviors of process I and II follow Arrhenius law. Process I (at 77 â 170 K) of all enzymes under all conditions are marked by low activation energy (Eá´) and small frequency factors which indicate small normal modes of vibration and inefficiency of the quenching process. Process II (at 170 â 200 K) has the same temperature dependence as the beta fluctuations of the protein hydration shell, indicating that the dynamic motions of proteins are slaved to the hydration shell, but with lower rate. Process III (at > 200 K) is explained by Vogel-Fulcher-Tamman (VFT) law and follows the motions of bulk solvent (viscosity) with smaller magnitude. The relaxation processes of W99, where the tryptophan residue is at the protein surface, differs from the other enzymes. W99 suffers quenching process much greater in process II and III compared to other enzymes, likely due to tighter coupling with solvent dynamics. Those results show that tryptophan phosphorescence is useful as a probe for monitoring molecular mobility with specific site information.
In order to improve the accuracy of the method, several different cryosolvents (glycerol water 75% v/v, glycerol water 90% v/v, ethylene glycol water 75% v/v, propylene glycol water 75% v/v, and aqueous sucrose and trehalose solutions) were used to measure the natural radiative decay rate of tryptophan, tyrosine, and their derivatives N-acetyl-tryptophanamide (NATA) and N-acetyl-tyrosinamide (NTyrA) at liquid nitrogen temperature (77 K). Different phosphorescence lifetimes at 77 K obtained from tyrosine, tryptophan and their derivatives in the different cryosolvents could reflect either their sensitivity to the solvent medium or residual matrix mobility at 77K. The effects of those factors need to be confirmed by observing phosphorescence decay kinetics at much lower temperature (4 K) to get clearer understanding of what causes the variation of lifetime in different solvents.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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