Document Type

Honors Project

Publication Date

5-29-2013

Abstract

Ribonuclease inhibitor (RI) is an intracellular mammalian protein which binds vertebrate-specific ribonucleases; this interaction is one of the tightest non-covalent interactions yet discovered. The biological activity of RI is poorly understood, but it is thought to regulate the biological functions of ribonucleases, which include initiating blood vessel growth, maintaining neuron viability, attacking pathogens, and mediating cell stress responses. RI is also involved in pathways unrelated to ribonucleases, including interactions with Drosha and PTEN, an anti-tumor protein.

One of the defining characteristics of RI is its oxidation sensitivity, a result of its unusually high cysteine content. The oxidation of RI is all-or-none and leads to complete loss of inhibitory activity; this feature is known to be relevant to at least some of its functions within the cell. However, the importance of these cysteines in the structure, inhibitory activity, and oxidation sensitivity of RI is unknown.

Variants of RI have been created in which sets of cysteines in RI have been conservatively replaced with other amino acids. In this study, we sought to recombinantly express and purify both native RI and the RI variants. Once pure, the variants were characterized for structural stability, inhibitory activity, oxidation sensitivity, and structural similarity to wild-type RI. Preliminary data suggest that the replacement of cysteines leads to an increase in structural stability but a decrease in inhibitory activity. This is a sign that tight binding of ribonucleases and vulnerability to oxidation may have co-evolved.

Level of Honors

magna cum laude

Department

Biochemistry

Advisor

Kimberly Dickson