A Study of the Impact of pH on the Structure and Stability of Epithelial Cadherin

Abadie, Catherine (2015) A Study of the Impact of pH on the Structure and Stability of Epithelial Cadherin. Undergraduate thesis, under the direction of Susan Pedigo from Chemistry and Biochemistry, The University of Mississippi.

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Abstract

Epithelial (E-) cadherin is a transmembrane protein whose function lies in cell-to-cell adhesion. It is a transmembrane component of adherens junctions of epithelial tissues, thereby connecting the actin cytoskeletons of adhered cells. It has five extracellular domains, a small transmembrane portion, and a cytoplasmic tail. The molecule must bind calcium in order to form an adhesive dimer with a cadherin molecule emanating from an adjacent cell. Cancer of the epithelial tissue, carcinoma, is accompanied by acidification of the tissue and a loss in cell adhesion. The long-term goal of this project is to determine the effect of a decrease in pH on the stability and function of adherens junctions. Toward that end, these experiments investigate the effects of pH on the thermal stability of the first two extracellular domains of E-cadherin (ECAD12). Due to E-cadherin’s anionic character, we hypothesize that a drop in the pH of the extracellular environment would stabilize the protein in its apo-state, while simultaneously decreasing its ability to bind calcium. Additionally, based on results on the effect of salt and decreased pH on dimerization by N-cadherin, we would expect that the level of dimer would increase as surface charges are neutralized either by protonation at lower pH or addition of salt. We used circular dichroism (CD) spectroscopy to study the stability of the protein as we decreased the pH from 7.4 to 5.5. Contrary to what we originally expected, our results indicated that protonating the residues in this range did not have a noticeable effect on the protein’s stability. However, studies conducted on the salt-dependence of stability at physiological pH found that electrostatic repulsion plays a small role in stability. From these studies of the pH dependence of stability, we conclude that, either electrostatic repulsion by charged amino acids have only a minor effect on protein stability or that relevant residues have pKa values that are outside of a physiologically relevant range. A preliminary study of the effect of salt on the formation of dimer was also performed using a kinetically-trapped mutant of E-cadherin. These chromatographic studies showed a distinct increase in the level of dimer in the presence of salt confirming our hypothesis that electrostatic repulsion plays a role in attenuating the affinity of the adhesive dimer in classical cadherins.

Item Type: Thesis (Undergraduate)
Creators: Abadie, Catherine
Student's Degree Program(s): B.A. in Biochemistry, B.A. in Arabic
Thesis Advisor: Susan Pedigo
Thesis Advisor's Department: Chemistry and Biochemistry
Institution: The University of Mississippi
Subjects: Q Science > QD Chemistry
Depositing User: Catherine Abadie
Date Deposited: 07 May 2015 15:57
Last Modified: 23 Oct 2015 19:12
URI: http://thesis.honors.olemiss.edu/id/eprint/362

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