NADH Conformation Assessed using Spectral Phasor Analysis: Illustrating Concepts from Molecular Folding to Metabolic Monitoring

Abstract

The wide applicability of spectroscopy makes fluorescence measurements on biomolecules an attractive modality for introducing biophysical topics into undergraduate physics laboratory courses. Here we show how the spectroscopic assessment of reduced nicotinamide adenine dinucleotide (NADH) conformation is useful for conveying concepts spanning multiple size scales. While advanced sources of optical contrast for NADH conformation include fluorescence excitation transfer efficiency, excited-state lifetime, and anisotropy decay rate, our recently published studies have shown how the quantification of autofluorescence spectrum shape using spectral phasor analysis is useful for monitoring NADH conformations during metabolic transitions. Here we present the pedagogical use of spectral phasor approaches to assess NADH conformation, with educational modules illustrating concepts ranging from molecular folding to protein binding, in addition to cellular metabolic monitoring. In the first module, we explore the NADH folding-unfolding transition in solution during methanol denaturation. The two-state nature of the transition and the degree of unfolding are evaluated using spectral phasors. A two-state solvent-denaturation model is used to estimate the free energy of transition. Next, we consider the protein binding of NADH to various dehydrogenases, showing that binding to different proteins can be discriminated optically, providing a spectroscopic means of examining ligand-binding behavior. The NADH binding constant to malate dehydrogenase is also measured, with the degree of ligand binding quantified using spectral phasors. Finally at the cellular level, the response of energy metabolism in Saccharomyces cerevisiae to various functional modifiers is monitored. We note that all modules were originally research projects involving undergraduate students, and so results here illustrate the relationship between student-driven, faculty-mentored research and the translation of those outcomes to courses centered around discovery-oriented learning.