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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
Structural changes induced by acidic pH in human apolipoprotein B-100.
José A Fernández-Higuero1,2, Asier Benito-Vicente1,2, Aitor Etxebarria1,2
1Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, UPV/EHU, Spain, Apdo. 644, 48080 Bilbao, Spain.
Lowers in LDL receptor (LDLR) pH induce structural changes in apolipoprotein B100 (apoB100), facilitating LDL release and degradation. These findings reveal dynamic LDL/LDLR interactions in endosomal environments.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Endosomal acidification triggers low-density lipoprotein (LDL) release from the LDL receptor (LDLR).
- While LDLR conformational changes are known, LDL particle structural changes remain less understood.
- Apolipoprotein B100 (apoB100) is the primary protein component of LDL.
Purpose of the Study:
- To investigate structural alterations in apoB100 under varying pH conditions.
- To examine changes in LDL size and morphology at physiological and endosomal pH.
- To elucidate the role of apoB100 structural changes in LDL/LDLR dissociation.
Main Methods:
- Infrared spectroscopy (IR) to analyze apoB100 secondary structure.
- Dynamic light scattering (DLS) to assess LDL particle size.
- Electron microscopy (EM) to visualize LDL morphology.
Main Results:
- IR spectroscopy revealed significant, reversible structural rearrangement of apoB100 upon pH decrease from 7.4 to 5.0.
- Acidification induced a reduction in beta-sheet content and an increase in alpha-helix structures within apoB100.
- DLS and EM showed no significant changes in LDL size or morphology.
- Observed apoB100 structural changes likely contribute to LDL release and subsequent degradation.
Conclusions:
- LDL/LDLR dissociation is more dynamic than previously thought.
- Endosomal acidification induces specific structural changes in apoB100, impacting LDL stability.
- These findings offer new insights into LDL/LDLR interactions within the endosomal low-pH environment.
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