Human apolipoprotein E (apoE) is 34-kDa 299 residue exchangeable apolipoprotein that plays a critical role in lipid transport and cholesterol metabolism in the plasma and brain. The APOE gene polymorphism results in three different alleles ϵ2, ϵ3, and ϵ4, which produce the common protein isoforms, apoE2, apoE3, and apoE4, respectively. Whereas apoE3 is considered to be the anti-atherogenic protein, apoE4 is considered a risk factor for developing Alzheimer’s disease (AD). ApoE associates mainly with VLDL but is associated with HDL-like particles in the brain. During brain cholesterol metabolism, apoE associates with HDL to form discoidal nascent HDL. In this study, we aim to determine the conformation of apoE4 by using chemical crosslinking, N-(1-pyrene)maleimide as a fluorescence probe, and mass spectrometry. Single cysteine mutants of apoE4 were expressed in E. coli, purified by affinity chromatography, and reconstituted with POPC. Discoidal rHDL (apoE4/POPC) was chemically cross-linked using a Cys-specific crosslinker. SDS-PAGE revealed only monomeric bands in all single-Cys variants. The lack of dimers suggests that the Cys residues on the apoE4 mutant molecules were not at a crosslinkable distance when bound to rHDL. We confirmed the conformation of apoE4 around the discoidal particles by an independent approach using spatially–sensitive fluorescence probes that allow us to measure the proximity of two Cys residues on two different apoE4 molecules on rHDL. The combination of cross-linker distance constraints and the excimer fluorescence emission spectra of the seven apoE4 mutants on HDL allows to conclude rule out a parallel double belt confirmation for apoE4 on discoidal rHDL. Our result support an out of sync parallel double-belt, anti-parallel double-belt, or a head to head hairpin model for apoE4/rHDL. The significance of this study is that it offers an innovative approach to obtain insight into the structure and organization of apoE on large lipoprotein complexes. Further, it allows us to identify potential differences between apoE3 and apoE4 from a structural perspective and determine distinguishing features that contribute to the role of apoE4 in developing AD.
|Commitee:||Lee, Yuan Yu, McAbee, Douglas D.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 58/01M(E), Masters Abstracts International|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be