Protein Science zyxwvutsrqponm (1996), zyxwvutsrq 5: 1874- 1882. Cambridge University zyxwvuts Press. Printed in the USA Copyright zyxwvutsrqpo 0 1996 The Protein Society High- and low-temperature unfolding of human high-density apolipoprotein A-2 OLGA GURSKY AND DAVID ATKINSON Department of Biophysics, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 021 18 (RECEIVED May 14, 1996; ACCEFTED June 25, 1996) 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 demon- strate 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 Th zyxwvutsrq = 56 zyxwvutsrqp 2 2 "C and vant Hoff's enthalpy AH(Th) = 17 2 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the a-helical structure. This suggests low-cooperativity apoA-2 unfold- ing. The apparent free energy of apoA-2 stabilization inferred from the CD analysis of the thermal unfolding, AG,,,,(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 Na,HPO, 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 AG,pp(250), 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 AH(T,) and low apparent ACp - 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. Keywords: circular dichroism; cold denaturation; differential scanning calorimetry; molten globule state; protein hydration Human plasma apolipoproteins A-1 (apoA-1, 28 kD, 243 residues) and A-2 (apoA-2, 17.4 kD, a dimer of two 77-residue molecules S-S linked via the Cys 6 pair) are the major proteins of the high- density lipoproteins (HDL) (Atkinson and Small, 1986). Concen- trations of plasma HDL and apoA-l correlate inversely with atherosclerotic coronary heart disease (Castelli et al., 1977). HDL particles that contain only apoA-l or both apoA-1 and apoA-2 have distinct metabolic properties. In contrast to apoA-I, which confers resistance to atherosclerosis, apoA-2 is likely to contribute to atherogenesis (Warden et al., 1993). ApoA-1 on HDL mediates reverse cholesterol transport and metabolism and provides a co- factor forthe enzyme 1ecithin:cholesterol acyltransferase (Fielding and Fielding, 1981). ApoA-2 has been inferred to importantly influence the HDL structural states, enhance HDL stability, and regulate the kinetics of apoA-1 transfer (reviewed by Scanu et al., 1982; Hedrick and Lusis, 1994). In contrast to apoA-1, which readily transfers among HDL particles in the course of their me- tabolism, apoA-2 is more strongly associated with HDL and has Reprintrequeststo:Olga Gursky, Department of Biophysics,Boston University School of Medicine, 80 East Concord Street, Boston, Massa- chusetts 02 11 8; e-mail: gursky@rned-biophd.bu.edu. been shown to displace apoA-1 from HDL (Lagocki and Scanu, 1980). The transfer of apoA-l and apoA-2 among HDL probably occurs via water-soluble lipid-poor apolipoprotein species (Os- borne et al., 1986). The sequences of apoA-1, apoA-2, and other exchangeable apo- lipoproteins contain characteristic 22-residue tandem repeats pre- dicted to form amphipathic a-helixes (McLachlan, 1977) that are proposed to play a key role in the lipid binding (Segrest et al., 1992). Far-UV CD spectra of these and other exchangeable apo- lipoproteins indicate high (35-60%) a-helical content that may increase substantially upon lipid binding (Atkinson and Small, 1986). Long amphipathic a-helixes, packed in elongated bundles, have been observed in the X-ray crystal structures of the locust apolipophorin Lp-111 (Breiter et al., 1991) and the N-terminal domain of human apolipoprotein E-3 (Wilson et al., 1991). Unhinging of the a-helix bundle has been proposed to facilitate apolipoprotein- lipid interactions (Breiter et al., 1991). To evaluate possible mechanisms of in vivo apolipoprotein trans- fer and to understand the molecular basis for the conformational adaptability of the exchangeable apolipoproteins to both aque- ous and lipid environment, we carried out a thermodynamic and structural analysis of lipid-free apoA-1 and apoA-2 under near- 1874