Testing A Model: Ca2+ Induced Exposure of Tryptophan in Neural-Cadherins

Author

Chinelo Udemgba

Date of Award

2009

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Susan Pedigo

Relational Format

Dissertation/Thesis

Abstract

Cadherins are transmembrane cell adhesion proteins that are critical for tissue formation and maintenance. Cell adhesion by cadherin requires binding of 3 calcium ions at the interfaces between the ectodomain modules. Recent data suggest a model in which calcium induces a relatively large conformational change in the first ectodomain module, domain 1, by causing the detachment of the A-strand from the core of domain 1 and exposure of a tryptophan in the second position, W2. The exposure of W2 is crucial for formation of a strand-crossover structure that is believed to be the adhesive dimer interface. In order to establish direct experimental evidence for this model, a construct of Neural-cadherin comprised of the first two ectodomain modules, NCAD12, and its single tryptophan mutants, W2A and W113 A, were examined. Neither mutation significantly affected the stability of the protein. Mutant proteins still bind calcium, although W113A showed less stabilization by calcium to thermal denaturation than the wild-type or W2A. The calcium-dependent characteristics of the proteins were analyzed separately with fluorescence spectroscopy and size exclusion chromatography. Regardless of the calcium level in solution, W2A was monomeric. W113A behaved like the wild type protein in that 1) there was dimer formed that was not in exchange with monomer, and 2) calcium allowed exchange between the monomer and dimer. We envisioned that a calcium-induced red shift in the fluorescence emission spectrum would confirm exposure of W2, however, there was no observable shift in the fluorescence signal with the wildtype or W113A. It is possible that the "open" conformation, in which W2 is exposed, is IV transient and difficult to monitor. We are currently exploring other mutations of W113 with the wild-type calcium binding properties. V

Recommended Citation

Udemgba, Chinelo, "Testing A Model: Ca2+ Induced Exposure of Tryptophan in Neural-Cadherins" (2009). Honors Theses. 2140.
https://egrove.olemiss.edu/hon_thesis/2140

Accessibility Status

Searchable text