NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption.
NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption.
复制标题
通过时间分辨荧光和双光子吸收揭示 NAD(P)H 结合构型。
DOI：
10.1016/j.bpj.2023.02.014
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发表时间：
2023
影响因子：
3.4
通讯作者：
Blacker TS
中科院分区：
文献类型：
--
作者：
Blacker TS
NADH和NADPH在调节代谢中起关键作用。它们的内源性荧光对酶结合很敏感，允许使用荧光寿命成像显微镜（FLIM）来确定细胞代谢状态的变化。然而，为了充分揭示潜在的生物化学，它们的荧光和结合动力学之间的关系需要更多的了解。在这里，我们通过时间和偏振分辨荧光和偏振双光子吸收测量来实现这一点。NADH与乳酸脱氢酶和NADPH与异柠檬酸脱氢酶的结合产生了两个生命周期。复合荧光各向异性表明，较短（1.3-1.6ns）的衰减成分伴随着烟酰胺环的局部运动，表明仅通过腺嘌呤部分附着。对于较长的寿命（3.2-4.4ns），烟酰胺的构象自由被完全限制。由于完全和部分烟酰胺结合是脱氢酶催化过程中公认的步骤，我们的研究结果统一了NADH和NADPH结合的光物理、结构和功能方面，并阐明了它们在细胞内寿命差异背后的生化过程。
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.