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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Amarasingha Ekanayaka, Sandamali"],"institution":null,"degree_name":"Ph.D.","degree_level":"Open Access Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Tamara L. Hendrickson"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013-01-01T08:00:00Z","date_published":"2013-01-01T08:00:00Z","updated_at":"2026-07-24T05:59:26Z","subjects":["GPI anchoring","GPI transamidase","In vitro assays for GPI-T","Transmembrane proteins","Biochemistry","Chemistry"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.wayne.edu/oa_dissertations/746","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Tamara L. Hendrickson"]},{"key":"dc:creator","label":"Author","values":["Amarasingha Ekanayaka, Sandamali"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2013-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Open Access Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["GPI anchoring","GPI transamidase","In vitro assays for GPI-T","Transmembrane proteins","Biochemistry","Chemistry"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.wayne.edu/oa_dissertations/746"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T"]}]}],"canonical_facts":{"dc:contributor":["Tamara L. Hendrickson"],"dc:creator":["Amarasingha Ekanayaka, Sandamali"],"dc:date.available":["2013-01-01T08:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.wayne.edu/oa_dissertations/746"],"dc:subject":["GPI anchoring","GPI transamidase","In vitro assays for GPI-T","Transmembrane proteins","Biochemistry","Chemistry"],"dc:title":["Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Open Access Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T05:59:26Z"}