The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

Biochemistry. 2010 Nov 9;49(44):9533-41. doi: 10.1021/bi101407m.

Abstract

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association−dissociation reaction of ApoE can be modeled with a monomer−dimer−tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association−dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer−dimer−tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alzheimer Disease / metabolism
  • Apolipoprotein E2 / chemistry
  • Apolipoprotein E2 / metabolism
  • Apolipoprotein E3 / chemistry
  • Apolipoprotein E3 / metabolism
  • Apolipoprotein E4 / chemistry
  • Apolipoprotein E4 / metabolism
  • Apolipoproteins E / chemistry*
  • Apolipoproteins E / metabolism
  • Fluorescence Resonance Energy Transfer* / methods
  • Humans
  • Kinetics
  • Protein Conformation
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Protein Multimerization
  • Spectrometry, Fluorescence / methods

Substances

  • Apolipoprotein E2
  • Apolipoprotein E3
  • Apolipoprotein E4
  • Apolipoproteins E
  • Protein Isoforms