Thermal unfolding of human high-density apolipoprotein A-1: Implications for a lipid-free molten globular state

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximate to 7, by using differential scanning calorimetry and circular dichroism, At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint T-m = 57 +/- 1 degrees C, calorimetric enthalpy Delta H(T-m) = 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy Delta H-v(T-m) approximate to 32.5 kcal/mol, and cooperativity Delta H-v(T-m)/Delta H(T-m) approximate to 0.16. The enthalpy Delta H(T-m) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximate to 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximate to 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.