Apolipoprotein E is an exchangeable apolipoprotein whose isoforms are associated with various disease risk profiles. Individuals bearing the APOE ϵ4 allele are at increased risk for developing Alzheimer’s disease compared to those bearing the APOE ϵ3 allele. The two isoforms differ in amino acid at position 112: apoE3 bears a Cys while apoE4 bears an Arg. It is hypothesized that the Cys to Arg substitution in apoE4 causes a decrease in stability in comparison to apoE3, which is exaggerated at endosomal pH <6.0. In our study, changes in secondary structure were monitored using circular dichroism at pH 7.4 and pH 3.5. Chemical denaturation indicated that both apoE3 and apoE4 retained their helical secondary structure at the lower pH, with a biphasic and monophasic guanidine HCl denaturation profile, respectively. Tertiary structure was monitored at both pH’s through fluorescence spectral characteristics and mobility of a fluorescent probe attached to each of the 7 major amphipathic α-helices of apoE3 and apoE4. The data showed decreases in fluorescence emission (FE), changes in fluorescence polarization (FP), and fluctuations in probe mobility, which were interpreted as likely formation of a molten globule. Formation of a molten globule appeared to occur during denaturation primarily for apoE4, and thermodynamic parameters of apoE4 showed a lower stability than apoE3, with a larger effect of pH. Taken together, our results suggest that the acidic pH in the endosomal compartments could interact with the native structure of apoE4 to generate a molten globule state that is able to bind endosomal membranes, other proteins, or itself. This study offers mechanistic insight into the impact of the single residue difference between apoE3 and apoE4 with regard to folding/unfolding behavior, and with regard to its physiological and pathological implications.
|Commitee:||Fraser, Deborah, Weers, Paul M.M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 57/01M(E), Masters Abstracts International|
|Keywords:||APOE, Dentaturation, Protein structure|
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