Circular dichroic properties and conformation of thionicotinamide dinucleotides

Area of Science:

• Biochemistry
• Spectroscopy
• Molecular Biology

Background:

• Nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) are crucial coenzymes in cellular metabolism.
• Understanding the conformational properties of NAD/NADH analogues is vital for elucidating their biological functions.
• Thioamide analogues (sNAD and sNADH) offer a unique probe for studying these properties.

Purpose of the Study:

• To investigate the conformational properties of 3-thioamide analogues of NAD and NADH (sNAD and sNADH).
• To analyze the influence of solvent environments (phosphate buffer pH 7 and ethanol) on their spectroscopic characteristics.
• To differentiate between conformational changes and solvent effects on spectroscopic signals.

Main Methods:

• Ultraviolet (UV) absorption spectroscopy.
• Circular Dichroism (CD) spectroscopy.
• Enzymatic cleavage of coenzymes followed by spectroscopic analysis of resulting fragments (AMP and sNMN(H)).

Main Results:

• A distinct couplet around 260 nm was observed for sNAD, absent in a mixture of AMP and sNMN, suggesting aromatic moiety stacking.
• Hyperchromism in the UV absorption band (~260 nm) was noted for both sNAD and sNADH.
• Spectroscopic data allowed differentiation of bands sensitive to conformational changes (open-stacking equilibrium) versus solvent effects.

Conclusions:

• The spectroscopic data strongly suggest an intramolecular stacking interaction between aromatic moieties in sNAD.
• The observed hyperchromism in sNAD and sNADH supports the presence of these stacking interactions.
• This study provides insights into the conformational dynamics of NAD/NADH analogues and their sensitivity to different solvent environments.