Date of Graduation

8-2018

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Paul Adams

Committee Member

Colin Heyes

Second Committee Member

Suresh Thallapuranam

Third Committee Member

Ralph Henry

Keywords

Ras, Rheb, TSC2, Tuberous Sclerosis

Abstract

Protein-protein interactions are vital in maintaining proper function and homeostasis in cells. Some signaling pathways are regulated by G-proteins that work like switches to activate and deactivate pathways. Mutations in these proteins, their effectors or the interaction between proteins may cause dysregulation of signals that can lead to many diseases.

Rheb, Ras homology enriched in brain, is a Ras family GTPase that is vital in regulation of the mTOR (mammalian target of rapamycin) pathway that signals cell proliferation and growth. Due to the low intrinsic GTPase activity of Rheb, a GTPase activating protein (GAP), Tuberous Sclerosis Complex 2 (TSC2) down regulates Rheb by enhancing its GTPase activity. Currently, very little information is available about TSC2 structures and the molecular details of the interaction between the two proteins. We explored various biochemical and biophysical information of Rheb-TSC2 interaction. In addition, we characterized the stability of the variant, TSC2-218 (D74A), using a 218 amino acid truncated construct of TSC2, and the effects of the single point mutation on the interactions with Rheb. In comparison to the WT, the D74A variant showed to maintain protein stability (thermal and chemical), an increase in secondary (alpha helical) structure, binding and GAP activity towards Rheb.

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