title: Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.
abstract: Apolipoprotein E (apoE) plays a critical role in cholesterol transport in both peripheral circulation and brain. Human apoE is a polymorphic 299-residue protein in which the less common E4 isoform differs from the major E3 isoform only by a C112R substitution. ApoE4 interacts with lipoprotein particles and with the amyloid-β peptide, and it is associated with increased incidence of cardiovascular and Alzheimer's disease. To understand the structural basis for the differences between apoE3 and E4 functionality, we used hydrogen-deuterium exchange coupled with a fragment separation method and mass spectrometric analysis to compare their secondary structures at near amino acid resolution. We determined the positions, dynamics, and stabilities of the helical segments in these two proteins, in their normal tetrameric state and in mutation-induced monomeric mutants. Consistent with prior X-ray crystallography and NMR results, the N-terminal domain contains four α-helices, 20 to 30 amino acids long. The C-terminal domain is relatively unstructured in the monomeric state but forms an α-helix ∼70 residues long in the self-associated tetrameric state. Helix stabilities are relatively low, 4 kcal/mol to 5 kcal/mol, consistent with flexibility and facile reversible unfolding. Secondary structure in the tetrameric apoE3 and E4 isoforms is similar except that some helical segments in apoE4 spanning residues 12 to 20 and 204 to 210 are unfolded. These conformational differences result from the C112R substitution in the N-terminal helix bundle and likely relate to a reduced ability of apoE4 to form tetramers, thereby increasing the concentration of functional apoE4 monomers, which gives rise to its higher lipid binding compared with apoE3.
authors.affiliation: Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104-5158.
authors.forename: Palaniappan S
authors.initials: PS
authors.lastname: Chetty
authors.affiliation: The Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Leland
authors.initials: L
authors.lastname: Mayne
authors.affiliation: Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104-5158.
authors.forename: Sissel
authors.initials: S
authors.lastname: Lund-Katz
authors.affiliation: The Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 engl@mail.med.upenn.edu mcp3@mail.med.upenn.edu.
authors.forename: S Walter
authors.identifier: 0000-0002-4802-2175
authors.initials: SW
authors.lastname: Englander
authors.affiliation: Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104-5158; engl@mail.med.upenn.edu mcp3@mail.med.upenn.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
mesh_terms: D019943:Amino Acid Substitution; D053318:Apolipoprotein E3 / Q000737:chemistry*; D053327:Apolipoprotein E4 / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D020022:Genetic Predisposition to Disease; D006859:Hydrogen / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008074:Lipoproteins / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D020125:Mutation, Missense; D017354:Point Mutation; D011485:Protein Binding; D000072417:Protein Domains; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D008074:Lipoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D006859:Hydrogen
keywords: amphipathic helix; apolipoprotein E; cholesterol; hydrogen exchange mass spectrometry; protein secondary structure