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Distinct NAD(P)H binding configurations revealed by fluorescence lifetime, anisotropy and polarised two-photon absorption

Blacker, Thomas S; Duchen, Michael R; Bain, Angus J; (2023) Distinct NAD(P)H binding configurations revealed by fluorescence lifetime, anisotropy and polarised two-photon absorption. Biophysical Journal 10.1016/j.bpj.2023.02.014. (In press). Green open access

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Abstract

NADH and NADPH play key roles in the regulation of metabolism. Their endogenous fluorescence is sensitive to enzyme binding, allowing changes in cellular metabolic state to be determined using fluorescence lifetime imaging microscopy (FLIM). However, to fully uncover the underlying biochemistry, the relationships between their fluorescence and binding dynamics require greater understanding. Here we accomplish this through time- and polarisation-resolved fluorescence and polarised two-photon absorption measurements. Two lifetimes result from binding of both NADH to lactate dehydrogenase and NADPH to isocitrate dehydrogenase. The composite fluorescence anisotropy indicates the shorter (1.3-1.6ns) decay component to be accompanied by local motion of the nicotinamide ring, pointing to attachment solely via the adenine moiety. For the longer lifetime (3.2-4.4ns), the nicotinamide conformational freedom is found to be fully restricted. As full and partial nicotinamide binding are recognised steps in dehydrogenase catalysis, our results unify photophysical, structural and functional aspects of NADH and NADPH binding and clarify the biochemical processes that underlie their contrasting intracellular lifetimes.

Type: Article
Title: Distinct NAD(P)H binding configurations revealed by fluorescence lifetime, anisotropy and polarised two-photon absorption
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.bpj.2023.02.014
Publisher version: https://doi.org/10.1016/j.bpj.2023.02.014
Language: English
Additional information: This work is licensed under an Attribution 4.0 International License (CC BY 4.0).
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy
URI: https://discovery.ucl.ac.uk/id/eprint/10165347
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