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Comparison of Mono-and Bifunctional Forms of Haemophilus influenzae TyrA Protein

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Comparison of Mono-and Bifunctional Forms of Haemophilus influenzae TyrA Protein

Kim, Hyun Young (2013) Comparison of Mono-and Bifunctional Forms of Haemophilus influenzae TyrA Protein. Masters thesis, Concordia University.

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Abstract

The TyrA protein family of dehydrogenases includes prephenate dehydrogenase (PD) which is dedicated to the synthesis of L-tyrosine (L-Tyr). PD is a monofunctional enzyme in many bacteria, but in Haemophilus influenzae, PD is bifunctional and is fused to chorismate mutase (CM). The present work is focused on understanding the importance of the CM domain in PD structure and stability in the presence of guanidine hydrochloride (Gdn-HCl) and variable temperatures (VT). Towards this goal, both full-length (FL) protein and an engineered monofunctional variant (∆80 CM-PD) form of TyrA from H. influenzae were overexpressed and purified using nickel affinity chromatography. Measurements by far-UV circular dichroism (CD) and steady-state fluorescence spectroscopies suggested that both proteins were completely unfolded by 4.2 M Gdn-HCl, although in the presence of 1.5 M to 2.4 M Gdn-HCl, the FL protein was more prone than ∆80 CM-PD to time-dependent protein precipitation. Thermal denaturation studies using far-UV CD spectroscopy and intrinsic fluorescence measurements revealed similar melting temperature of ~52°C for both proteins. VT- FTIR experiments showed that the temperature-mediated unfolding pathways of the two enzymes were very similar but not identical. Additionally the active site ligands, NAD+ and L-Tyr, stabilized intramolecular β-sheets of both proteins by ~5ᵒC. VT experiments also support the idea that H. influenzae TyrA proteins retain their dimeric nature during unfolding and highlight the importance of the dimer interface in TyrA structure and function.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Kim, Hyun Young
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:19 April 2013
Thesis Supervisor(s):Turnbull, Joanne
ID Code:977149
Deposited By: HYUN YOUNG KIM
Deposited On:13 Jun 2013 19:47
Last Modified:18 Jan 2018 17:43
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