HIGH-TEMPERATURE AND LOW-TEMPERATURE UNFOLDING OF HUMAN HIGH-DENSITY APOLIPOPROTEIN A-2

Human plasma apolipoprotein A-2 (apoA-2) is the second major protein o f the high-density lipoproteins that mediate the transport and metabol ism of cholesterol. Using CD spectroscopy and differential scanning ca lorimetry, we demonstrate that the structure of lipid-free apoA-2 in n eutral low-salt solutions is most stable at similar to 25 degrees C an d unfolds reversibly both upon heating and cooling from 25 degrees C. High-temperature unfolding of apoA-2, monitored by far-UV CD, extends from 25-85 degrees C with midpoint T-h = 56 +/- 2 degrees C and vant H off's enthalpy Delta H(T-h) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-helical struc ture. This suggests low-cooperativity apoA-2 unfolding. The apparent f ree energy of apoA-2 stabilization inferred from the CD analysis of th e thermal unfolding, Delta G(app)(25 degrees) = 0.82 +/- 0.15 kcal/mol , agrees with the value determined from chemical denaturation. Enhance d low-temperature stability of apoA-2 observed upon increase in Na2HPO 4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may b e linked to reduced water activity. The close proximity of the heat an d cold unfolding transitions, that is consistent with low Delta G(app) (25 degrees), indicates that lipid-free apoA-2 has a substantial hydro phobic core but is only marginally stable under near-physiological sol vent conditions. This suggests that in vivo apoA-2 transfer is unlikel y to proceed via the lipid-free state. Low Delta H(T-h) and low appare nt Delta C-p similar to 0.52 kcal/mol . K inferred from the far-UV CD analysis of apoA-2 unfolding, and absence of tertiary packing interact ions involving Tyr groups suggested by near-UV CD, are consistent with a molten globular-like state of lipid-free apoA-2.