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Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage
Insights
Apolipoprotein C-III-1 fragments were studied for lipid binding. The C-terminal fragment (apoLP-C-III-B) binds phospholipids via an amphipathic alpha helix, highlighting hydrophobicity
Area of Science:
- Biochemistry
- Structural Biology
- Lipid Metabolism
Background:
- Apolipoprotein C-III (apoLP-C-III) plays a role in lipid metabolism.
- Understanding the structural basis of apoLP-C-III's lipid binding is crucial.
Purpose of the Study:
- To investigate the lipid binding properties of apoLP-C-III fragments.
- To determine the structural requirements for phospholipid binding by apoLP-C-III.
Main Methods:
- Thrombin cleavage of apoLP-C-III-1 into fragments.
- Circular dichroism and fluorescence spectroscopy for structural analysis.
- Cesium chloride density gradient ultracentrifugation for complex isolation.
Main Results:
- ApoLP-C-III-1 fragment (residues 1-40) showed disordered structure and no lipid binding.
- ApoLP-C-III-1 fragment (residues 41-79) exhibited conformational changes and formed peptide-phospholipid complexes.
- Complexes had a lipid to protein molar ratio of 12:1, indicating significant lipid binding capacity.
Conclusions:
- An amphipathic alpha helix with distinct polar and nonpolar faces is the fundamental unit for phospholipid binding by plasma apolipoproteins.
- The hydrophobicity of the nonpolar face of the alpha helix is critical for effective lipid binding.
Abstract:
We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.