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Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains.

J R Wetterau1, L P Aggerbeck, S C Rall

  • 1Gladstone Foundation Laboratories for Cardiovascular Disease, University of California, San Francisco 94140-0608.

The Journal of Biological Chemistry
|May 5, 1988
PubMed
Summary

Human apolipoprotein E3 (apoE) has two distinct structural domains. These domains exhibit different stabilities, with the amino-terminal domain being more stable than the carboxyl-terminal domain.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Chemistry

Background:

  • Human apolipoprotein E (apoE) plays a crucial role in lipid metabolism and transport.
  • Understanding the structural stability of apoE is essential for elucidating its function in various physiological and pathological processes.

Purpose of the Study:

  • To investigate the structural stability and domain organization of human apolipoprotein E3 (apoE) in aqueous solution.
  • To identify and characterize the distinct structural domains within apoE and assess their individual stabilities.

Main Methods:

  • Guanidine hydrochloride (GdnHCl) denaturation monitored by circular dichroism (CD) spectroscopy.
  • Limited proteolysis using five different enzymes to identify proteolytically resistant regions.
  • Analysis of thrombolytic fragments as models for apoE domains.

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Main Results:

  • GdnHCl denaturation revealed a biphasic unfolding curve with two transition midpoints (0.7 and 2.5 M GdnHCl), indicating stable intermediate structures.
  • Limited proteolysis identified two proteolytically resistant domains: an amino-terminal (residues 20-165) and a carboxyl-terminal (residues 225-299).
  • Denaturation of amino-terminal and carboxyl-terminal fragments yielded transition midpoints of 2.4 M and 0.7 M GdnHCl, respectively, with free energies of 8-12 kcal/mol and ~4 kcal/mol.

Conclusions:

  • The two domains identified by limited proteolysis directly correspond to the domains detected by denaturation experiments.
  • Human apoE3 possesses two independently folded structural domains with significantly different stabilities in aqueous solution.
  • The amino-terminal domain is considerably more stable than the carboxyl-terminal domain, suggesting a unique structural characteristic among apolipoproteins.