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Analysis of conformational behaviour exhibited by dihydrofolate reductase during catalysis

Hughes, Robert 2018. Analysis of conformational behaviour exhibited by dihydrofolate reductase during catalysis. PhD Thesis, Cardiff University.
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Abstract

Protein motions, which occur on a multitude of timescales, are known to be central to enzyme catalysis. However, between enzyme variants there exists diversity in the influence such motions have. Understanding why apparently similar enzymes that have similar primary sequences, tertiary structures and rate constants utilize different motions is therefore of great interest. Dihydrofolate reductase (DHFR) has been used extensively as a model to study such relationships between protein motions and catalysis. Firstly, we have probed conformational behaviour exhibited by different DHFR variants by a simplified, cost-effective NMR based approach utilizing selective 13C labelling of methionine and tryptophan sidechains. Before this work, DHFR from Escherichia coli (EcDHFR) was the only known DHFR to adopt an occluded conformation following the chemical step. 13C labelling of methionine and tryptophan sidechains is shown to be sufficient to probe such conformational behaviour in EcDHFR, with clear chemical shift perturbations observed between the two conformational states. No such chemical shift perturbations are observed in spectra relating to DHFR from Thermotoga maritima (TmDHFR) or DHFR from Moritella profunda (MpDHFR), where an occluded conformation is not adopted following the chemical step. Through amino acid sequence analysis DHFR from Salmonella enterica (SeDHFR) was identified as conserving a key hydrogen bonding interaction known to stabilise an occluded conformation in EcDHFR. By analysing chemical shift perturbations of 13C labelled methionine and tryptophan sidechains, it has been confirmed SeDHFR exhibits similar conformational behaviour to EcDHFR. The second part of this work aimed to further explore the contribution of femtosecond dynamics acting upon the chemical step of catalysis in SeDHFR. The role of such motions coupling to the chemical step is hugely controversial. Our work, with SeDHFR, aligns with previous reports that the influence of dynamic coupling to the chemical step is minimised in catalysis by DHFR. To further explore the role of dynamic coupling to the chemical step in other enzyme families, L-lactate dehydrogenase-1 from Staphylococcus aureus (SaLDH) was studied. Initial findings align with work done with DHFR variants and reports the dynamic coupling to the chemical step is minimised at close to physiological conditions.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Date of First Compliant Deposit: 23 May 2019
Last Modified: 04 Aug 2022 02:06
URI: https://orca.cardiff.ac.uk/id/eprint/122816

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