title: Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4.
abstract: Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site- directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indi- cating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Mizuguchi
authors.forename: Mami
authors.initials: M
authors.lastname: Hata
authors.forename: Padmaja
authors.initials: P
authors.lastname: Dhanasekaran
authors.forename: Margaret
authors.initials: M
authors.lastname: Nickel
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Okuhira
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
authors.forename: Sissel
authors.initials: S
authors.lastname: Lund-Katz
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000378:metabolism*; D053327:Apolipoprotein E4 / Q000737:chemistry* / Q000378:metabolism*; D002645:Chickens; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000378:metabolism; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D008055:Lipids; D010713:Phosphatidylcholines; D020033:Protein Isoforms; D011721:Pyrenes; D053835:Unilamellar Liposomes; C038518:acrylodan; D014364:Tryptophan; C030984:pyrene; D015081:2-Naphthylamine; D019791:Guanidine