Figure 1.

Two conformations of NADH marked as “Unfolded” and “Folded” alongside the temperature dependence of ³¹P NMR results for 30 mM NADH in pure deuterated solvents: DMSO-d₆ and D₂O. (A) Below the dashed line: 1D NMR spectra of 30 mM NADH in pure D₂O at temperatures ranging from 310 to 353 K, demonstrating line broadening with increasing temperature. Above the dashed line: 1D NMR spectra of 30 mM NADH in pure DMSO-d₆ over the same temperature range, exhibiting line narrowing as temperature increases. (B) Line widths for NADH as a function of temperature, displaying a monotonic increase in pure D₂O and a monotonic decrease in pure DMSO-d₆. (C) Spin–lattice relaxation rate (R₁) as a function of temperature, showing a monotonic decrease in pure D₂O and a local maximum in pure DMSO-d₆. (D) Spin–spin relaxation rate (R₂) as a function of temperature, following the same trend as the line widths in (B): a monotonic increase in D₂O and a monotonic decrease in DMSO-d₆.