Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism $ Prathima Acharya a,1,2 , Mark L. Segall b,2 , Mohamed Zaiou b , Julie Morrow c , Karl H. Weisgraber c , Michael C. Phillips b , Sissel Lund-Katz b , Julian Snow a, * a Department of Chemistry and Biochemistry, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA b Joseph Stokes Jr. Research Institute, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA c Gladstone Institutes of Cardiovascular Disease and Neurological Disease, University of California, San Francisco, CA 94141-9100, USA Received 26 March 2002; accepted 26 June 2002 Abstract Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4 < apoE3 < apoE2; T max c 52 < 56 < 59 jC, respectively). Helical contents at the onset and completion of denaturation (as monitored calorimetrically) were about 51% and 35%, respectively, for 22-kDa apoE3. As much as 70% of the unfolding enthalpy for 22-kDa apoE3 could be due to disruption of a-helix. The monomeric 22-kDa fragments unfold differently, with both apoE3 and apoE4 exhibiting unfolding intermediates, whereas apoE2 unfolds according to a two-state mechanism. Interactions with the C-terminal domain appear to destabilize the N-terminal domains in full-length apoE2 and apoE3, but less so for full-length apoE4. Self-association of full-length apoE at higher concentrations (1.5 – 2.5 mg/ml), as well as interaction of full-length apoE with phospholipid, enhances thermal stability. Cooperative changes in secondary structure, as monitored by circular dichroism, begin near 37 jC for full-length apoE3 and apoE4, indicating that full-length apoE3 and apoE4, but not apoE2, may be partially unfolded in vivo. The differences in stability and unfolding behavior are likely to contribute to the molecular basis for defects in lipid transport and neurological function induced by apoE polymorphism. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Apolipoprotein E; Differential scanning calorimetry; Circular dichroism 1. Introduction ApoE, first identified as a component of very low-density lipoproteins (VLDL), is associated with several classes of serum lipoproteins, including high-density lipoproteins (HDL) and chylomicron remnants [1]. As a ligand for cell-surface receptors of the low-density lipoprotein (LDL) family and cell-surface proteoglycans, apoE plays a key role in cholesterol transport and lipoprotein metabolism. In humans, there are three allele products, designated apoE2, apoE3 and apoE4, with apoE3 being the most common isoform. The isoforms differ at residues 112 and 158; whereas apoE3 has cysteine at position 112 and arginine at 158, apoE2 has cysteine and apoE4 arginine at both positions. The functional consequences of these point muta- 1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1388-1981(02)00263-9 Abbreviations: apo, apolipoprotein; CD, circular dichroism; DSC, differential scanning calorimetry; HDL, high-density lipoprotein; LDL, low-density lipoprotein; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- phocholine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Trx, thioredoxin; VLDL, very low-density lipoprotein $ This work was supported by National Institutes of Health Grants HL56083 and NS35939. * Corresponding author. Tel.: +1-215-596-8837; fax: +1-215-596-8543. E-mail addresses: phillipsmi@email.chop.edu (M.C. Phillips), j.snow@usip.edu (J. Snow). 1 Present address: Diosynth RTP, Inc., 3000 Weston Pkwy, Cary, NC 27513, USA. 2 These two authors contributed equally to the work. www.bba-direct.com Biochimica et Biophysica Acta 1584 (2002) 9 – 19