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Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Published on: August 15, 2014
The structure of apolipoprotein A-II in discoidal high density lipoproteins
R A Gangani D Silva1, Lumelle A Schneeweis2, Srinivasan C Krishnan3
1Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45237.
Insights
Researchers developed a structural model for apolipoprotein A-II (apoA-II) in high density lipoproteins (HDL). This model, supported by cross-linking/mass spectrometry and infrared spectroscopy, reveals an antiparallel arrangement of apoA-II within HDL particles.
Area of Science:
- Biochemistry
- Structural Biology
- Cardiovascular Research
Background:
- High levels of high density lipoproteins (HDL) are known to reduce atherosclerosis risk.
- Apolipoprotein A-II (apoA-II) is a major protein component of HDL, but its structure within HDL is not well understood.
- Existing structural models primarily focus on apolipoprotein A-I (apoA-I).
Purpose of the Study:
- To present a detailed structural model of apoA-II within reconstituted HDL (rHDL) particles.
- To elucidate the molecular arrangement of apoA-II in HDL using experimental data.
- To provide a basis for understanding apoA-II structure in more complex HDL.
Main Methods:
- Utilized chemical cross-linking combined with mass spectrometry (MS) to identify proximal lysine residues.
- Employed tandem mass spectrometry (MS/MS) for confirmation of cross-linked peptides.
- Applied internal reflection infrared spectroscopy to determine the molecular arrangement of apoA-II.
Main Results:
- Identified and confirmed 14 cross-links within apoA-II in rHDL.
- Infrared spectroscopy suggested a belt-like arrangement of apoA-II wrapping around the rHDL disc.
- Structural analysis refuted a parallel arrangement and supported two antiparallel models, particularly a "double hairpin" conformation.
Conclusions:
- The study presents the first detailed structural model for apoA-II in rHDL.
- The findings indicate an antiparallel arrangement of apoA-II, with a "double hairpin" model being most supported.
- This structural insight is crucial for understanding apoA-II's role in HDL and its association with cardiovascular health.
Abstract:
It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.
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