Honors Theses

Date of Award

2014

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Susan Pedigo

Relational Format

Dissertation/Thesis

Abstract

Cadherin is a major mediator of cell adhesion whose adhesive function is dependent upon calcium binding. However, the binding of calcium is not completely understood. The binding of calcium can be monitored using an abbreviated construct that contains only the first two extracellular domains (EC12) which includes two tryptophan residues, W2 and W113. Single tryptophan mutants were created which contain only W113 (W2A) and W2 (W113F). Fluorescence spectra were obtained of W2A, W113F, and WT in the presence and absence of calcium. Calcium titration data were collected for each protein. In both experiments, calcium binding increased the fluorescence signal of W113F and decreased the signal of W2. The apparent Kd values from titration experiments showed a much lower affinity for calcium binding in the WT construct than in the single-tryptophan mutants. Simulations of calcium binding data address this apparent discrepancy. Two models were used to create simulations: an equal and independent model and an unequal and independent model. W2 signal, W113 signal, and the sum of the two, WT, were represented in the simulations. The effect of span, offset, and Kd values were tested for the simulated W2 and W113 data to observe the effects on the summed data. Changes in offset of the two mutants had only a trivial effect. Span dictates the span of the sum, and also affects the Kd value. When simulating an increasing W113 signal and decreasing W2 signal, the Kd of the sum shifted to greater values. When both signals have the same direction, the sum is represented by an intermediate Kd. Therefore, the Kd of the sum is dictated by the span, Kd, and direction of the signal of W2 and W113. This indicates that W2A and W113F are good reporters of the two separate classes of sites in EC12 constructs of N-cadherin.

Included in

Chemistry Commons

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