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 approximately 25 degrees C and 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 Th = 56 +/- 2 degrees C and vant Hoff's enthalpy delta H(Th) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-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, delta G(app)(25 degrees) = 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 delta G(app)(25 degrees), 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 delta H(Th) and low apparent delta Cp approximately 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.
Gursky
O
O
Department of Biophysics, Boston University School of Medicine, Massachusetts 02118, USA. gursky@med-biophd.bu.edu
Atkinson
D
D
D016633:Apolipoprotein A-II / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003080:Cold Temperature; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D016633:Apolipoprotein A-II; D010710:Phosphates; D005990:Glycerol; C018279:sodium phosphate