Date of Award

Fall 2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Steven A. Jones

Abstract

Tensin is a large "docking" protein found in the adhesive junctions of animal cells and recruited early in the development of cell-substrate contacts. There it binds to the cytoplasmic domain of integrin β1 and caps the barbed ends of filamentous actin. This forms a rational basis for its implication in a direct role in the mechanics of membrane-cytoskeleton interactions. Tensin provides a physical link between the actin cytoskeleton, integrins, and other proteins at the cell-substrate contacts. Its overall biochemical properties are a function of its domain composition and architecture, i.e., the domains that are present and their relative positions in the molecule, and specific details of amino acid sequence and post-translational modifications. Tensin can be used as an investigative tool to help explain the physiology of cell-substrate contacts at the molecular scale. The C2 domain indicated to be present at the N-terminus of tensin, along with the phosphatase domain, bears a close homology to PTEN (Phosphatase and Tensin Homolog), a well known tumor suppressor. The main research objective was to study the structural features of the C2 domain of human tensin as well as to investigate its thermal stability.

For this purpose, the C2 domain gene, after being synthesized by Polymerase Chain Reaction, was cloned into the engineered pET-14b vector that would also encode a hexa-Histidine tag. The C2 domain gene was then overexpressed in E.coli and the inclusion bodies thus formed were solubilized and lysed. Ni-NTA metal affinity chromatography was performed to obtain the purified C2 domain. Circular dichroism was used for the initial study of the structural features of the purified C2 domain. After determining that the purified C2 domain was unstable, refolding attempts were done. Later studies of the domain by circular dichroism in the far-UV and near-UV wavelengths indicated that the domain unfolded gradually with increasing amounts of the denaturant GuHCl and also that it retained some amount of tertiary structure prior to denaturation.

The absence of an endothermic peak in the Differential Scanning Calorimetry experiments only suggests the need for further extensive refolding methods not attempted in this work and might also indicate the need for the presence of a phosphatase domain at its N-terminus for thermodynamic stability. The results of this work suggest the presence of a C2 domain in human tensin, which has not been documented previously. Determination of all the structural and functional characteristics of the C2 domain in the long run will contribute to the understanding of the role of tensin in tumor suppression and cell signaling.

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