{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/87260"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/87260","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2","abstract":"The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.","abstract_html":"The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.","abstract_has_math":false,"creators":["Chen, Ci-Di"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Molecular and Integrative Physiology","degree_department":null,"school":null,"contributors":["Byron Kemper"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-28T15:50:16Z","date_published":"2015-09-28T15:50:16Z","updated_at":"2026-07-22T22:26:28Z","subjects":["Biology, Molecular"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI9834662"],"render_values":[{"text":"(MiAaPQ)AAI9834662","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/87260","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Byron Kemper"]},{"key":"dc:creator","label":"Author","values":["Chen, Ci-Di"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-28T15:50:16Z","10000-01-01","1998"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Molecular and Integrative Physiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Molecular"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/87260","(MiAaPQ)AAI9834662"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.","Made available in DSpace on 2015-09-28T15:50:16Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9834662.pdf: 3517201 bytes, checksum: aa0ca4c0afe50962dde521dd630040c9 (MD5) Previous issue date: 1998","Embargo set by: Seth Robbins for item 88541 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","89 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998."]},{"key":"dc:title","label":"Title","values":["Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2"]}]}],"canonical_facts":{"dc:contributor":["Byron Kemper"],"dc:creator":["Chen, Ci-Di"],"dc:date":["2015-09-28T15:50:16Z","10000-01-01","1998"],"dc:description":["The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.","Made available in DSpace on 2015-09-28T15:50:16Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9834662.pdf: 3517201 bytes, checksum: aa0ca4c0afe50962dde521dd630040c9 (MD5) Previous issue date: 1998","Embargo set by: Seth Robbins for item 88541 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","89 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998."],"dc:identifier":["http://hdl.handle.net/2142/87260","(MiAaPQ)AAI9834662"],"dc:language":["eng"],"dc:subject":["Biology, Molecular"],"dc:title":["Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2"],"dc:type":["text"],"thesis:degree_discipline":["Molecular and Integrative Physiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:28Z"}