Effect of organic co-solvents on the NMR chemical shifts and dynamics of dihydrofolate reductase from E. coli

Cosgrave, Stella 2018. Effect of organic co-solvents on the NMR chemical shifts and dynamics of dihydrofolate reductase from E. coli. MPhil Thesis, Cardiff University. Item availability restricted.

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Abstract

Enzymes are large biological molecules that can exist in many different states and each state may have multiple conformations. The probability of finding an enzyme in a particular state is governed by the free energy landscape and the energy barriers between the different states. The energy landscape of an enzyme can be perturbed through making changes to the external conditions such as the reaction medium, which can cause a shift in the delicate equilibrium between states. Experimental evidence suggests that the model for environmentally coupled tunnelling is inconsistent for catalysis by dihydrofolate reductase (DHFR) from E coli.. It is proposed that catalysis is governed by electrostatics and characterised by populations of sub-states with distinct conformations and kinetics. Here, using solution NMR spectroscopy, the enzyme is probed at the point of the chemical reaction step by subjecting a mimic of the Michaelis complex (EcDHFR: NADP+: folate) to organic co-solvents in order to perturb the energy landscape of the protein by altering the viscosity and dielectric constant of the enzymatic reaction medium. The chemical shift assignment of the Michaelis complex mimic under standard buffer conditions, in the presence of 17% methanol and 17 % glycerol co-solvents (to effect almost isodielectric mediums that differ in viscosity) is reported. The majority of atoms in the protein complex in the presence of 17% glycerol and 17% methanol show small chemical shift perturbations (Chapter 4.0). The ps-ns relaxation dynamics of the Michaelis complex mimic were measured via solution NMR at 600 MHz and 900 MHz in the presence of co-solvents to determine any alterations in the timescale of dynamics on this timescale. The results indicate slightly increased mobility of some residues in loop regions in the presence of methanol and glycerol co-solvents and slightly decreased mobility of some residues in areas of defined secondary structure (Chapter 5.0).

Item Type:	Thesis (MPhil)
Date Type:	Submission
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
Date of First Compliant Deposit:	5 December 2018
Last Modified:	06 Dec 2018 10:23
URI:	https://orca.cardiff.ac.uk/id/eprint/117386

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