Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Tamara L. Hendrickson

Abstract

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE

By

SANDAMALI AMARASINGHA EKANAYAKA

December 2013

Advisor: Dr. Tamara L. Hendrickson

Major: Biochemistry

Degree: Doctor of Philosophy

The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for GPI-T will facilitate innumerable new experiments to study this complicated enzyme. The three core subunits of GPI-T (Gpi8, Gpi16, and Gaa1) were co-purified from a GPI8 knockout Saccharomyces cerevisiae strain containing a plasmid that expresses Gpi8 with an appended glutathione-S-transferase (GST) domain. Peptide substrates for GPI-T were synthesized and modified to contain a pair of chromophores suitable for the development of a fluorescence resonance energy transfer (FRET) assay. GPI-T activity was observed as a time-dependent increase in fluorescence by incubating peptides with pure, solubilized GPI-T in the presence of hydroxylamine, a small GPI anchor mimic. A FRET assay was developed and optimized to monitor GPI anchoring activity in vitro. The assay was used to investigate various aspects of GPI-T, including the importance of the C-terminal hydrophobic region in peptide substrates, the identity of the residue at the site of modification, substrate selectivity, and the effect of cofactors, co-substrates and inhibitors for GPI-T .To date no one has demonstrated robust GPI-T activity with pure solubilized GPI-T. Thus, this new FRET assay represents the first high-throughput method to quantitatively analyze GPI-T activity in vitro.

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