Honors Theses
Date of Award
2015
Document Type
Undergraduate Thesis
Department
Chemistry and Biochemistry
First Advisor
Susan Pedigo
Relational Format
Dissertation/Thesis
Abstract
Cadherins are a family of cell-surface glycoproteins that aid in cell adhesion between two proximal cells. The characteristic cadherin extracellular structure consists of five ~100 amino acid domains connected by seven-residue linker regions. Cadherins mediate adhesion via adherens junctions by forming a strand-swapped structure between identical protomers from apposing cells. Upon calcium binding, the swapping of the N- terminal strands between protomers allows for the symmetrical docking of a conserved tryptophan residue in the N-terminal strand into the hydrophobic pocket of its partner protomer. The two most-well-studied members of the classical cadherin family are Neural- (N-) and Epithelial- (E-) cadherin. Although these two proteins are very similar in sequence, structure and function, they display a striking difference in the calcium- dependent kinetics of dimerization. Given that the adhesive interface is in domain 1, we are investigating the difference between domains 1 of E-cadherin (ECAD1) and N- cadherin (NCAD1). Mouse ECAD1 was cloned by inserting a stop codon after the coding sequence for domain 1. Recombinant protein was expressed in E.coli and purified using His-Tag chromatography. Purity of the ECAD1 stock was assessed by SDS- PAGE. The stock was characterized spectrally using UV-vis and fluorescence spectroscopy, indicating that ECAD1 was folded. Denaturant-induced unfolding studies indicated that ECAD1 was significantly lower in stability in comparison to NCAD1. Next the presence of dimer in stocks of ECAD1 and NCAD1 was assessed using Size Exclusion Chromatography (SEC). NCAD1 formed a kinetically trapped dimer while ECAD1 was found to form monomer exclusively. In summary, our results indicate that the difference in kinetics of dimerization may be due to a difference in the intrinsic stability of domain 1.
Recommended Citation
Edmondson, Molly, "Characterization of Epithelial Cadherin Domain 1" (2015). Honors Theses. 715.
https://egrove.olemiss.edu/hon_thesis/715
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