Structural differences between apolipoprotein E3 and E4 as measured by ¹⁹F NMR

Abstract

Abstract:The apolipoprotein E family contains three major isoforms (ApoE4, E3, and E2) that are directly involved with lipoprotein metabolism and cholesterol transport. ApoE3 and apoE4 differ in only a single amino acid with an arginine in apoE4 changed to a cysteine at position 112 in apoE3. Yet only apoE4 is recognized as a risk factor for Alzheimer's disease. Here we used 19 F NMR to examine structural differences between apoE4 and apoE3 and the effect of the C-terminal domain on the N-terminal domain. After incorporation of 5-19 F-tryptophan the 1D 19 F NMR spectra were compared for the N-terminal domain and for the full length proteins. The NMR spectra of the N-terminal region (residues 1-191) are reasonably well resolved while those of the full length wild-type proteins are broad and ill-defined suggesting considerable conformational heterogeneity. At least four of the seven tryptophan residues in the wild type protein appear to be solvent exposed. NMR spectra of the wild-type proteins were compared to apoE containing four mutations in the C-terminal region that gives rise to a monomeric form either of apoE3 under native conditions (Zhang et al., Biochemistry 2007; 46: 10722-10732) or apoE4 in the presence of 1 M urea. For either wild-type or mutant proteins the differences in tryptophan resonances in the Nterminal region of the protein suggest structural differences between apoE3 and apoE4. We conclude that these differences occur both as a consequence of the Arg158Cys mutation and as a consequence of the interaction with the C-terminal domain.

Mass Spectrometry-Based Protein Footprinting Characterizes the Structures of Oligomeric Apolipoprotein E2, E3, and E4

“…W39 cannot be detected as modified, although the reporting tryptic peptide 39-61 is of low abundance. A similar conclusion was reached by Garai et al (24) using apoE labeled with 19 F tryptophan. Our conclusion arises from a comparison of the FPOP yields for same amino-acid residue located throughout the same protein.…”

“…ApoE2, ApoE3, ApoE4, and ApoE3MM, were expressed in E. coli as described elsewhere (24). The proteins were dissolved in 6 M guanidinium chloride and dialyzed overnight into phosphate buffered saline (PBS) solution containing 100 μM TCEP disulfide reductant.…”

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

“…This agrees with the NMR structure of the monomeric apoE3 demonstrating that several residues in this region are involved in the formation of hydrogen bonds and salt-bridges with the N-terminal domain [25, 27]. In addition, 19 F NMR spectra of 5- 19 F-Trp incorporated into apoE3 and apoE4 demonstrated the structural differences in the N-terminal domain as a consequence of interaction with the C-terminal domain [52]. Therefore, the N-terminal domain serves as a folding template for the C-terminal domain [27], and thus, the less pronounced stabilizing domain-domain interactions in apoE4 would lead to the less organized conformation of the C-terminal domain, resulting in the different self-association behaviors through the C-terminal domain between apoE3 and apoE4 in solution [20].…”

The Binding of Apolipoprotein E to Oligomers and Fibrils of Amyloid-β Alters the Kinetics of Amyloid Aggregation

“…Recombinant lipid-free apoE was prepared according to the protocol described previously. 27 Lipidation of recombinant apoE was performed by incubating 30 μM apoE with 2 mg/mL small unilamellar vesicles (SUV) of 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) in 20 mM phosphate, pH 7.4 buffer containing 150 mM NaCl and 5 mM β-mercaptoethanol (βME). 28 The solution was incubated at 25 °C overnight.…”