Open Access Dissertation
Date of Award
Tamara L. Hendrickson
DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE
SANDAMALI AMARASINGHA EKANAYAKA
Advisor: Dr. Tamara L. Hendrickson
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.
Amarasingha Ekanayaka, Sandamali, "Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T" (2013). Wayne State University Dissertations. 746.