Conformation of NADH studied by fluorescence excitation transfer spectroscopy

Dihydronicotinamide adenine dinucleotide (NADH), is known to stack in two limiting conformations. Surprisingly, previous experimental work on NADH has not clearly defined whether this folding and unfolding process can be described as first order (involving only two states) or whether one or more intermediates must be included in the description. In addition, a large disparity exists between reported equilibrium constants for the aqueous solution at room temperature. Using methanol as a denaturant, we have used fluorescence excitation transfer spectroscopy to probe the stacking/un-stacking equilibrium. Our results can be represented using a simple two state model. Furthermore, the mole fraction of aqueous NADH in the stacked configuration is significantly higher than previously reported. Using a thermodynamic two state model, we have determined deltaG(o)(H2O) = 8.8 +/- 1.4 kJ mol(-1). From excitation energy transfer measurements the fraction of NADH in the folded form at 295 K was determined to be 0.55.