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

Abstract

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 μM 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.