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Title: Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles

Abstract

Further understanding of the structure and function of plasma apolipoproteins requires the determination of their high-resolution structures when complexed with lipids. In these studies, the production of homogeneous, biologically active lipoprotein particles of apolipoprotein E complexed with dipalmitoylphosphatidylcholine and their crystallization and X-ray diffraction are demonstrated. High-resolution structural information is available for several soluble plasma apolipoproteins (apos) in a lipid-free state. However, this information provides limited insight into structure–function relationships, as this class of proteins primarily performs its functions of lipid transport and modulation of lipid metabolism in a lipid-bound state on lipoprotein particles. Here, the possibility of generating homogeneous lipoprotein particles that could be crystallized was explored, opening the possibility of obtaining high-resolution structural information by X-ray crystallography. To test this possibility, apoE4 complexed with the phospholipid dipalmitoylphosphatidylcholine was chosen. Uniform particles containing 50% lipid and 50% apoE4 were obtained and crystallized using the hanging-drop method. Two crystal forms diffract to beyond 8 Å resolution.

Authors:
 [1];  [1];  [2];  [1];  [2];  [2]
  1. Gladstone Institutes of Cardiovascular and Neurological Disease, University of California, San Francisco, CA 94158 (United States)
  2. (United States)
Publication Date:
OSTI Identifier:
22356175
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 61; Journal Issue: Pt 11; Other Information: PMCID: PMC1978127; PMID: 16511213; PUBLISHER-ID: pu5108; OAI: oai:pubmedcentral.nih.gov:1978127; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; BOUND STATE; CRYSTALLIZATION; CRYSTALLOGRAPHY; CRYSTALS; MODULATION; PLASMA; RESOLUTION; X-RAY DIFFRACTION

Citation Formats

Newhouse, Yvonne, Peters-Libeu, Clare, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, Weisgraber, Karl H., E-mail: kweisgraber@gladstone.ucsf.edu, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, and Department of Pathology, University of California, San Francisco, CA 94158. Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles. United Kingdom: N. p., 2005. Web. doi:10.1107/S1744309105032410.
Newhouse, Yvonne, Peters-Libeu, Clare, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, Weisgraber, Karl H., E-mail: kweisgraber@gladstone.ucsf.edu, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, & Department of Pathology, University of California, San Francisco, CA 94158. Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles. United Kingdom. doi:10.1107/S1744309105032410.
Newhouse, Yvonne, Peters-Libeu, Clare, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, Weisgraber, Karl H., E-mail: kweisgraber@gladstone.ucsf.edu, Cardiovascular Research Institute, University of California, San Francisco, CA 94158, and Department of Pathology, University of California, San Francisco, CA 94158. Tue . "Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles". United Kingdom. doi:10.1107/S1744309105032410.
@article{osti_22356175,
title = {Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles},
author = {Newhouse, Yvonne and Peters-Libeu, Clare and Cardiovascular Research Institute, University of California, San Francisco, CA 94158 and Weisgraber, Karl H., E-mail: kweisgraber@gladstone.ucsf.edu and Cardiovascular Research Institute, University of California, San Francisco, CA 94158 and Department of Pathology, University of California, San Francisco, CA 94158},
abstractNote = {Further understanding of the structure and function of plasma apolipoproteins requires the determination of their high-resolution structures when complexed with lipids. In these studies, the production of homogeneous, biologically active lipoprotein particles of apolipoprotein E complexed with dipalmitoylphosphatidylcholine and their crystallization and X-ray diffraction are demonstrated. High-resolution structural information is available for several soluble plasma apolipoproteins (apos) in a lipid-free state. However, this information provides limited insight into structure–function relationships, as this class of proteins primarily performs its functions of lipid transport and modulation of lipid metabolism in a lipid-bound state on lipoprotein particles. Here, the possibility of generating homogeneous lipoprotein particles that could be crystallized was explored, opening the possibility of obtaining high-resolution structural information by X-ray crystallography. To test this possibility, apoE4 complexed with the phospholipid dipalmitoylphosphatidylcholine was chosen. Uniform particles containing 50% lipid and 50% apoE4 were obtained and crystallized using the hanging-drop method. Two crystal forms diffract to beyond 8 Å resolution.},
doi = {10.1107/S1744309105032410},
journal = {Acta Crystallographica. Section F},
number = Pt 11,
volume = 61,
place = {United Kingdom},
year = {Tue Nov 01 00:00:00 EST 2005},
month = {Tue Nov 01 00:00:00 EST 2005}
}
  • A lipoprotein implicated in mycobacterial cell-wall biosynthesis, LpqW, was expressed in E. coli. Crystals were obtained that diffracted to 2.4 Å resolution. Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acidmore » lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%(w/v) PEG 4000, 8–16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way.« less
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  • A recombinant virus-like particle that is a potential oral hepatitis E vaccine was crystallized. Diffraction data were collected to 8.3 Å resolution and the X-ray structure was phased with the aid of a low-resolution density map determined using cryo-electron microscopy data. Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3more » protein (residue 70–123) fused to the N-terminus of the ORF2 protein (residues 112–608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 Å resolution. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 337, b = 343, c = 346 Å, α = β = γ = 90°, and contain one particle per asymmetric unit.« less
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