High‐ and low‐temperature unfolding of human high‐density apolipoprotein A‐2

Human plasma apolipoprotein A‐2 (apoA‐2) is the second major protein of the high‐density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid‐free apoA‐2 in neutral low‐salt solutions is most stable at ∼25 °C and unfolds reversibly both upon heating and cooling from 25 °C. High‐temperature unfolding of apoA‐2, monitored by far‐UV CD, extends from 25–85 °C with midpoint T_h = 56 ± 2 °C and vant Hoff's enthalpy ΔH(T_h) = 17 ± 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the α‐helical structure. This suggests low‐cooperativity apoA‐2 unfolding. The apparent free energy of apoA‐2 stabilization inferred from the CD analysis of the thermal unfolding, ΔG_app(25°) = 0.82 ± 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low‐temperature stability of apoA‐2 observed upon increase in Na₂HPO₄ concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low ΔG_APP (25°), indicates that lipid‐free apoA‐2 has a substantial hydrophobic core but is only marginally stable under near‐physiological solvent conditions. This suggests that in vivo apoA‐2 transfer is unlikely to proceed via the lipid‐free state. Low ΔH(T_h) and low apparent ΔC_p ∼ 0.52 kcal/mol‐K inferred from the far‐UV CD analysis of apoA‐2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near‐UV CD, are consistent with a molten globular‐like state of lipid‐free apoA‐2.