title: Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.
abstract: Although a high-resolution X-ray structure for the N-terminal domain of apolipoprotein E (apoE) in the lipid-free state has been solved, our knowledge of the structure of full-length apoE in a lipid-bound state is limited to an X-ray model fitting a molecular envelope at 10-A resolution. To add molecular detail to the molecular envelope, we used cysteine mutagenesis to incorporate spin labels for analysis with electron paramagnetic resonance (EPR) spectroscopy. Twelve cysteine residues were introduced singly and in pairs at unique locations throughout apoE4 and labeled with an EPR spin probe. The labeled apoE4 was combined with dipalmitoylphosphatidylcholine, the particles were purified, and spectra were determined for 24 combinations (single and double) of the cysteine mutants. Data on the conformation, mobility, distance, and surface exposure of regions revealed by the cysteine probes were modeled into the molecular envelope of apoE bound to dipalmitoylphosphatidylcholine that had been determined by X-ray analysis. This EPR model of apoE in a native lipid-bound state validates the structural model derived from X-ray analysis and provides additional insight into apoE structure-function relationships.
authors.affiliation: Gladstone Institutes of Cardiovascular and Neurological Diseases, 1650 Owens Street, San Francisco, CA 94158, USA.
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Budamagunta
authors.forename: Yvonne N
authors.initials: YN
authors.lastname: Newhouse
authors.forename: Karl H
authors.initials: KH
authors.lastname: Weisgraber
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry* / Q000378:metabolism; D001057:Apolipoproteins E / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels
chemical_list: D001057:Apolipoproteins E; D008055:Lipids; D011994:Recombinant Proteins; D013113:Spin Labels; D015060:1,2-Dipalmitoylphosphatidylcholine; D003545:Cysteine