Date of Award
Doctor of Philosophy (PhD)
Steven A. Jones
Cell migration is a key characteristic of embryogenesis, inflammation, wound healing, tumor cell metastasis and a broad range of other normal and pathological processes. Accumulating evidence suggests that the protein tensin provides a physical link between transmembrane receptors, proteins commonly associated with signal transduction, and the actin cytoskeleton. Adhesion involves three broad classes of macromolecules: ExtraCellular Matrix (ECM) molecules, transmembrane adhesion receptors, and intracellular adhesion plaque proteins. Tensin is particularly enriched in Fibrillar Adhesions (FA's), though it is also present to a modest extent in Focal Contacts (FC's). Src Homology 2 (SH2) domains function in the transmission of molecular signals that start at the cell surface, pass through the plasma membrane, and engage the inner workings of the cell. SH2 domains carry out their function by binding with high affinity to phosphotyrosine-containing protein targets in a sequence-specific and largely phosphorylation-dependent manner. In this work, SH2 domain of protein tensin, a component of cell-substrate contacts with close connections to cancer is used as an investigative tool, to view the scientific problem from the perspective of a biophysicist.
Recombinant DNA technology was used to clone the SH2 gene and overexpress the recombinant SH2 domain. Molecular biology techniques like Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), immunblot analysis were used to identify and characterize the protein molecular mass. Circular Dichroism (CD) spectroscopy was used to determine the secondary structure of the protein and to demonstrate that the SH2 domain folds into a compact, stable molecule. Chemical denaturation and heat denaturation studies were done using Differential Scanning Calorimetry (DSC) to determine the thermostability of the SH2 domain. These studies reveal that the tensin-SH2 domain is highly stable as compared with some other known SH2 domains. Detailed knowledge of the structure and function of tensin will accelerate acquisition of more detailed knowledge of other focal adhesion components, advancing the development of molecular models of cell attachment and migration. Such knowledge is of interest to basic science as well as medicine. Moreover, it could also provide a model for nanotechnology development, providing inspiration for the design of novel types of molecular recognition and functionality, and materials design and fabrication.
Kulkarni, Yogesh Mohan, "" (2007). Dissertation. 501.