Cold Denaturation of Apo-Myoglobin in the Presence of Polyols

Thermal unfolding of apo-myoglobin (apoMb) in various polyols, ethylene glycol (EG), 1,2-butanediol (12BDO), 1,4-butanediol (14BDO), 1,2-hexanediol (12HDO), 1,6-hexanediol (16HDO), glycerol, and 1,2,6-hexanetriol (126HTO) was monitored by circular dichroism (CD) spectroscopy from -15°C to 90°C at 222 nm (data in file 01*.csv). Structural changes of apoMb were analyzed by following the protein's intrinsic fluorescence (files 02*.csv to 08*.csv). In addition, the fluorescence changes of N-acetyl tryptophan amide (NATA), a tryptophan analogue in all polyols (files 09*.csv to15*.csv) was measured as a control. The integrity of the hydrophobic core packing was probed using ANS fluorescence (files 17*.csv to 30*.csv). The thermodynamic parameters of cold and heat-induced unfolding were estimated (file 31*.csv).

Further, replica exchange molecular dynamics (REMD) simulations were performed on six protein-polyol-water systems: apoMb in water, and in 10% concentrations of EG, 12BDO, 12HDO, glycerol, and 126HTO. Simulations were conducted in three temperature ranges: (i) ambient (280–320 K) using native apoMb, (ii) cold (250–300 K) using unfolded apoMb, and (iii) heat (300–350 K) using unfolded apoMb (parameters in All_REMD_Parameters).