Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease.
The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.
Morrow
Julie A
JA
Gladstone Institutes of Cardiovascular Disease and Neurological Disease, San Francisco, California 94141-9100, USA.
Hatters
Danny M
DM
Lu
Bin
B
Hochtl
Peter
P
Oberg
Keith A
KA
Rupp
Bernhard
B
Weisgraber
Karl H
KH
D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000737:chemistry*; D004134:Dimyristoylphosphatidylcholine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea
D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D010446:Peptide Fragments; D020033:Protein Isoforms; D014508:Urea; D010434:Pepsin A; D004134:Dimyristoylphosphatidylcholine