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 i s critically involved in the transport and metabolism of cholesterol a nd in the pathogenesis of atherosclerosis. We reexamined the thermal u nfolding of lipid-free apoA-1 in low-salt solution at pH approximate t o 7, by using differential scanning calorimetry and circular dichroism , At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is r esolved as an extended peak (25 degrees C-90 degrees C) that can be la rgely accounted for by a single reversible non-two-state transition wi th 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 inferr ed by CD to constitute approximate to 60% of apoA-1 amino acids. Faran d near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary st ructure but a largely melted tertiary structure for apoA-1 at approxim ate to 37 degrees C and pH 7. This suggests a molten globular-like sta te for lipid-free apoA-1 under near-physiological conditions. Our resu lts suggest that in vivo lipid binding by apoA-1 may be mediated via t he molten globular apolipoprotein state in plasma.