Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T

<p>DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE</p> <p>By</p> <p>SANDAMALI AMARASINGHA EKANAYAKA</p> <p>December 2013</p> <p>Advisor: Dr. Tamara L. Hendrickson</p> <p>Major: Biochemistry</p> <p>Degree: Doctor of Philosophy</p> <p>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.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2013

Author dc:creator

• Amarasingha Ekanayaka, Sandamali

Contributors dc:contributor

• Tamara L. Hendrickson

• GPI anchoring
• GPI transamidase
• In vitro assays for GPI-T
• Transmembrane proteins
• Biochemistry
• Chemistry

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/746

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1745