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A rapid analysis of variations in conformational behavior during dihydrofolate reductase catalysis

Hughes, Robert L., Johnson, Luke A. ORCID:, Behiry, Enas M., Loveridge, Edric Joel and Allemann, Rudolf Konrad ORCID: 2017. A rapid analysis of variations in conformational behavior during dihydrofolate reductase catalysis. Biochemistry 56 (15) , pp. 2126-2133. 10.1021/acs.biochem.7b00045

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Protein flexibility is central to enzyme catalysis, yet it remains challenging both to predict conformational behavior on the basis of analysis of amino acid sequence and protein structure and to provide the necessary breadth of experimental support to any such predictions. Here a generic and rapid procedure for identifying conformational changes during dihydrofolate reductase (DHFR) catalysis is described. Using DHFR from Escherichia coli (EcDHFR), selective side-chain 13C labeling of methionine and tryptophan residues is shown to be sufficient to detect the closed-to-occluded conformational transition that follows the chemical step in the catalytic cycle, with clear chemical shift perturbations found for both methionine methyl and tryptophan indole groups. In contrast, no such perturbations are seen for the DHFR from the psychrophile Moritella profunda, where the equivalent conformational change is absent. Like EcDHFR, Salmonella enterica DHFR shows experimental evidence of a large-scale conformational change following hydride transfer that relies on conservation of a key hydrogen bonding interaction between the M20 and GH loops, directly comparable to the closed-to-occluded conformational change observed in EcDHFR. For the hyperthermophile Thermotoga maritima, no chemical shift perturbations were observed, suggesting that no major conformational change occurs during the catalytic cycle. In spite of their conserved tertiary structures, DHFRs display variations in conformational sampling that occurs concurrently with catalysis.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Publisher: American Chemical Society
ISSN: 0006-2960
Funders: BBSRC
Date of First Compliant Deposit: 15 June 2017
Date of Acceptance: 3 April 2017
Last Modified: 17 Nov 2023 02:25

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