title: Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation.
abstract: To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the Delta(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA. igorshko@bu.edu
authors.forename: Irina N
authors.initials: IN
authors.lastname: Gorshkova
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Horng-Yuan
authors.initials: HY
authors.lastname: Kan
authors.forename: Angeliki
authors.initials: A
authors.lastname: Chroni
authors.forename: Vassilis I
authors.initials: VI
authors.lastname: Zannis
authors.forename: David
authors.initials: D
authors.lastname: Atkinson
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008055:Lipids / Q000737:chemistry / Q000235:genetics; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008954:Models, Biological; D009154:Mutation / Q000187:drug effects; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014407:Tumor Cells, Cultured
chemical_list: D000817:Anilino Naphthalenesulfonates; D016632:Apolipoprotein A-I; D008055:Lipids; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D012996:Solutions; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D019791:Guanidine