Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

The two-component nature of the autofluorescence response is indicative of whether a single or several pathways are affected.

Phasor analysis on fluorescence signals is a sensitive approach for analyzing multicomponent systems. Initially developed for time-resolved measurements, a spectral version has been used for the rapid identification of regions during the spectral imaging of biological systems. Here we show that quantitative information regarding conformation can be obtained from phasor analysis of fluorescence spectrum shape. Methanol denaturation of NADH and NADH binding to various dehydrogenase proteins are used as model reactions. Thermodynamic constants are calculated and compared with previous studies based on more direct measures of conformation. Next, the quantitative monitoring of UV-excited autofluorescence spectrum shape during chemically-induced metabolic transitions is presented and discussed in terms of NADH-utilizing pathways. Results show how phasor analysis is useful in assessing two-state behavior, and in interpreting autofluorescence as emission from an ensemble of cellular NADH forms.

with high affinity and exhibit a robust open probability~0.4. Substituting dual alanines for the 'IQ' residues in the IQ element significantly weakens apocalmodulin binding, and markedly diminishes peak open probability (~0.1). Overexpressing apocalmodulin fully rescues open probability, demonstrating that the effects on opening reflect apocalmodulin binding per se. This extensive structural and functional similarity substantiates a striking conservation of calmodulin regulation across Nav and Cav channels, joint investigation of which now presents as a genuinely synergistic endeavor.

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