{"id":{"repo_id":"whiterose","oai_identifier":"oai:etheses.whiterose.ac.uk:1027"},"canonical_url":"https://search.dev.ndltd.org/etd/whiterose/oai:etheses.whiterose.ac.uk:1027","repository":{"repo_id":"whiterose","name":"White Rose University Consortium","base_url":"https://etheses.whiterose.ac.uk/cgi/oai2"},"display":{"title":"Structural analysis of LMO2 for the development of a small molecule inhibitor","abstract":"LMO2 is a nuclear LIM-only protein encoded by a gene located on chromosome 11p13 and was originally discovered through its activation due to specific chromosomal translocations in patients with T-cell acute lymphoblastic leukaemia (T-ALL). The specific chromosomal translocations occur with either the T-cell δ receptor gene (14q11) or T-cell β receptor gene (7q35) and result in aberrant LMO2 expression in T-cells. Transgenic mouse models of LMO2 induced T cell neoplasias showed that enforced LMO2 expression caused accumulation of immature thymic T cells, followed by clonal T cell tumours with long latency. LMO2 is therefore a specific therapeutic target as not only is it associated with chromosomal translocations but is also expressed in approximately 50% of T-ALL. The aims of this project were to structurally determine LMO2 for structure based drug development of small molecules that will target LMO2 protein-protein interactions. LMO2 could not be purified alone as removal of the fusion tag resulted in severe precipitation of the free LMO2. Consequently, LMO2 was co-expressed with an antibody single domain termed VH#576, and purified to a high yield and purity. A final construct of LMO2, spanning residues 9 to 147, bound to VH#576 has been crystallised and the structure solved, to a medium resolution of 3.3Å, using phase information from single anomalous dispersion (SAD) data in combination with molecular replacement. Using a mammalian two-hybrid mutagenesis screen, key VH#576 binding residues have been identified. This data can be used, in combination with the crystal structure of VH#576/LMO2ΔN7ΔC11, to produce a Pharmacophore model for in silico screening and lead drug discovery. In addition to the crystallography approach, NMR was also investigated as a means to collect structural data on VH#576, in solution. A protocol has been developed to isotopically label and purify VH#576 along with unlabeled LMO2, in order to increase the stability of the antibody single domain for NMR data acquisition. Solving the structure of VH#576 by NMR requires further data collection. Advantageously, NMR solution structures represent more physiological environment and comparison of a VH#576 NMR structure and the crystal structure would enable the detection of any crystallisation artifacts.","abstract_html":"LMO2 is a nuclear LIM-only protein encoded by a gene located on chromosome 11p13 and was originally discovered through its activation due to specific chromosomal translocations in patients with T-cell acute lymphoblastic leukaemia (T-ALL). The specific chromosomal translocations occur with either the T-cell δ receptor gene (14q11) or T-cell β receptor gene (7q35) and result in aberrant LMO2 expression in T-cells. Transgenic mouse models of LMO2 induced T cell neoplasias showed that enforced LMO2 expression caused accumulation of immature thymic T cells, followed by clonal T cell tumours with long latency. LMO2 is therefore a specific therapeutic target as not only is it associated with chromosomal translocations but is also expressed in approximately 50% of T-ALL. The aims of this project were to structurally determine LMO2 for structure based drug development of small molecules that will target LMO2 protein-protein interactions. LMO2 could not be purified alone as removal of the fusion tag resulted in severe precipitation of the free LMO2. Consequently, LMO2 was co-expressed with an antibody single domain termed VH#576, and purified to a high yield and purity. A final construct of LMO2, spanning residues 9 to 147, bound to VH#576 has been crystallised and the structure solved, to a medium resolution of 3.3Å, using phase information from single anomalous dispersion (SAD) data in combination with molecular replacement. Using a mammalian two-hybrid mutagenesis screen, key VH#576 binding residues have been identified. This data can be used, in combination with the crystal structure of VH#576/LMO2ΔN7ΔC11, to produce a Pharmacophore model for in silico screening and lead drug discovery. In addition to the crystallography approach, NMR was also investigated as a means to collect structural data on VH#576, in solution. A protocol has been developed to isotopically label and purify VH#576 along with unlabeled LMO2, in order to increase the stability of the antibody single domain for NMR data acquisition. Solving the structure of VH#576 by NMR requires further data collection. Advantageously, NMR solution structures represent more physiological environment and comparison of a VH#576 NMR structure and the crystal structure would enable the detection of any crystallisation artifacts.","abstract_has_math":false,"creators":["Sewell, Helen"],"institution":"University of Leeds","degree_name":"Ph.D","degree_level":"doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Rabbitts, T."],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-04","date_published":"2010-04","updated_at":"2026-07-24T06:03:54Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["uk.bl.ethos.524509"],"render_values":[{"text":"uk.bl.ethos.524509","href":null,"code":true}]}]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Rabbitts, T."]},{"key":"dc:creator","label":"Author","values":["Sewell, Helen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2010-04"]},{"key":"dc:date.issued","label":"Date","values":["2010-04"]},{"key":"dc:publisher.commercial","label":"Dc Publisher Commercial","values":["University of Leeds"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Institute of Molecular Medicine (LIMM) (Leeds)"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Leeds"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://etheses.whiterose.ac.uk/id/eprint/1027/"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Ph.D"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["uk.bl.ethos.524509"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://etheses.whiterose.ac.uk/id/eprint/1027/1/Sewell_H_LIMM_PhD_2010.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["LMO2 is a nuclear LIM-only protein encoded by a gene located on chromosome 11p13 and was originally discovered through its activation due to specific chromosomal translocations in patients with T-cell acute lymphoblastic leukaemia (T-ALL). The specific chromosomal translocations occur with either the T-cell δ receptor gene (14q11) or T-cell β receptor gene (7q35) and result in aberrant LMO2 expression in T-cells. Transgenic mouse models of LMO2 induced T cell neoplasias showed that enforced LMO2 expression caused accumulation of immature thymic T cells, followed by clonal T cell tumours with long latency. LMO2 is therefore a specific therapeutic target as not only is it associated with chromosomal translocations but is also expressed in approximately 50% of T-ALL. The aims of this project were to structurally determine LMO2 for structure based drug development of small molecules that will target LMO2 protein-protein interactions. LMO2 could not be purified alone as removal of the fusion tag resulted in severe precipitation of the free LMO2. Consequently, LMO2 was co-expressed with an antibody single domain termed VH#576, and purified to a high yield and purity. A final construct of LMO2, spanning residues 9 to 147, bound to VH#576 has been crystallised and the structure solved, to a medium resolution of 3.3Å, using phase information from single anomalous dispersion (SAD) data in combination with molecular replacement. Using a mammalian two-hybrid mutagenesis screen, key VH#576 binding residues have been identified. This data can be used, in combination with the crystal structure of VH#576/LMO2ΔN7ΔC11, to produce a Pharmacophore model for in silico screening and lead drug discovery. In addition to the crystallography approach, NMR was also investigated as a means to collect structural data on VH#576, in solution. A protocol has been developed to isotopically label and purify VH#576 along with unlabeled LMO2, in order to increase the stability of the antibody single domain for NMR data acquisition. Solving the structure of VH#576 by NMR requires further data collection. Advantageously, NMR solution structures represent more physiological environment and comparison of a VH#576 NMR structure and the crystal structure would enable the detection of any crystallisation artifacts."]},{"key":"dc:format","label":"Dc Format","values":["text"]},{"key":"dc:title","label":"Title","values":["Structural analysis of LMO2 for the development of a small molecule inhibitor"]}]}],"canonical_facts":{"dc:contributor.advisor":["Rabbitts, T."],"dc:creator":["Sewell, Helen"],"dc:date":["2010-04"],"dc:date.issued":["2010-04"],"dc:description.abstract":["LMO2 is a nuclear LIM-only protein encoded by a gene located on chromosome 11p13 and was originally discovered through its activation due to specific chromosomal translocations in patients with T-cell acute lymphoblastic leukaemia (T-ALL). The specific chromosomal translocations occur with either the T-cell δ receptor gene (14q11) or T-cell β receptor gene (7q35) and result in aberrant LMO2 expression in T-cells. Transgenic mouse models of LMO2 induced T cell neoplasias showed that enforced LMO2 expression caused accumulation of immature thymic T cells, followed by clonal T cell tumours with long latency. LMO2 is therefore a specific therapeutic target as not only is it associated with chromosomal translocations but is also expressed in approximately 50% of T-ALL. The aims of this project were to structurally determine LMO2 for structure based drug development of small molecules that will target LMO2 protein-protein interactions. LMO2 could not be purified alone as removal of the fusion tag resulted in severe precipitation of the free LMO2. Consequently, LMO2 was co-expressed with an antibody single domain termed VH#576, and purified to a high yield and purity. A final construct of LMO2, spanning residues 9 to 147, bound to VH#576 has been crystallised and the structure solved, to a medium resolution of 3.3Å, using phase information from single anomalous dispersion (SAD) data in combination with molecular replacement. Using a mammalian two-hybrid mutagenesis screen, key VH#576 binding residues have been identified. This data can be used, in combination with the crystal structure of VH#576/LMO2ΔN7ΔC11, to produce a Pharmacophore model for in silico screening and lead drug discovery. In addition to the crystallography approach, NMR was also investigated as a means to collect structural data on VH#576, in solution. A protocol has been developed to isotopically label and purify VH#576 along with unlabeled LMO2, in order to increase the stability of the antibody single domain for NMR data acquisition. Solving the structure of VH#576 by NMR requires further data collection. Advantageously, NMR solution structures represent more physiological environment and comparison of a VH#576 NMR structure and the crystal structure would enable the detection of any crystallisation artifacts."],"dc:format":["text"],"dc:identifier":["uk.bl.ethos.524509"],"dc:identifier.uri":["https://etheses.whiterose.ac.uk/id/eprint/1027/1/Sewell_H_LIMM_PhD_2010.pdf"],"dc:publisher.commercial":["University of Leeds"],"dc:publisher.department":["Institute of Molecular Medicine (LIMM) (Leeds)"],"dc:publisher.institution":["University of Leeds"],"dc:relation.isreferencedby":["https://etheses.whiterose.ac.uk/id/eprint/1027/"],"dc:title":["Structural analysis of LMO2 for the development of a small molecule inhibitor"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["doctoral"],"dc:type.qualificationname":["Ph.D"]},"updated_at":"2026-07-24T06:03:54Z"}