Date of Award
Chemistry and Biochemistry
Szabo, Arthur G.,
CC BY-NC-ND 4.0
Fluorescence spectroscopy of tryptophan residues is a valuable tool for dissecting the structure, function and dynamic behaviour of proteins. An important step in method development is the use of tryptophan analogues with unique photophysical properties that extend the utility of intrinsic protein fluorescence. Two difficulties limit the use of these analogues. Their structure, while similar to that of tryptophan, can perturb native protein structure resulting in misfolding or inactivity. There are also problems from an inability of tryptophanyl-tRNA synthetase (TrpRS) to use these analogues as a substrate in the tRNA charging reaction. The tryptophan residue in rat parvalbumin F102W is buried in the highly hydrophobic and conformationally restrained environment of the protein's core. Differences in hydrophobicity, size and hydrogen bonding capability of tryptophan analogues were expected to affect both the structure and stability of this protein once incorporated at position 102. All analogues were successfully incorporated. Incorporation of 5-hydroxytryptophan proved difficult and required an optimization of incubation temperature during induction. 7-azatryptophan and 5-hydroxytryptophan destabilized the protein while 4-fluorotryptophan, 5-fluorotryptophan and 6-fluorotryptophan stabilized the protein. Single tryptophan mutants of Bacillus stearothermophilis TrpRS showed large structural changes when forming the 4-fluorotryptophan-AMP intermediate. These changes were limited to the formation of the first intermediate. This is rationalized as a large global change occurring across both subunits that could affect substrate binding. H. sapien tryptophanyl-tRNA synthetase was expressed and found to be active when using tryptophan analogues as substrates. Differences in fluorescence properties of the intermediate suggest a unique environment at the active site.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2002 .A23. Source: Dissertation Abstracts International, Volume: 64-01, Section: B, page: 0183. Adviser: Arthur G. Szabo. Thesis (Ph.D.)--University of Windsor (Canada), 2002.
Acchione, Mauro., "Characterization of the effects of biosynthetically incorporated tryptophan analogues in rat parvalbumin F102W and structure-function studies of tryptophanyl-tRNA synthetase." (2002). Electronic Theses and Dissertations. 1408.