Dissertation/Thesis Abstract

The Conformation of Apolipoprotein E4 in Discoidal High-Density Lipoprotein by Crosslinking and Fluorescence Spectroscopy
by Bala, Noor S., M.S., California State University, Long Beach, 2018, 110; 10784272
Abstract (Summary)

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.

Indexing (document details)
Advisor: Narayanaswami, Vasanthy
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
Source Type: DISSERTATION
Subjects: Biochemistry
Keywords:
Publication Number: 10784272
ISBN: 9780438209008
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