{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/21067"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/21067","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"The anti-fluorescein antibody active site: A single-chain site-specific mutagenesis study","abstract":"Restriction data tranferred 2014-07-01T11:21:50-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","abstract_html":"Restriction data tranferred 2014-07-01T11:21:50-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","abstract_has_math":false,"creators":["Denzin, Lisa Kae"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Microbiology","degree_department":null,"school":null,"contributors":["Voss, Edward W., Jr."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:57:23Z","date_published":"2011-05-07T12:57:23Z","updated_at":"2026-07-22T22:25:17Z","subjects":["Biology, Microbiology","Health Sciences, Immunology"],"languages":["eng"],"rights":["Copyright 1992 Denzin, Lisa Kae"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9305505","(UMI)AAI9305505"],"render_values":[{"text":"AAI9305505","href":null,"code":true},{"text":"(UMI)AAI9305505","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/21067","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Voss, Edward W., Jr."]},{"key":"dc:creator","label":"Author","values":["Denzin, Lisa Kae"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:57:23Z","10000-01-01","1992"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"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, Microbiology","Health Sciences, Immunology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1992 Denzin, Lisa Kae"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9305505","(UMI)AAI9305505","http://hdl.handle.net/2142/21067"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Restriction data tranferred 2014-07-01T11:21:50-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only","Crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (K$\\sb{\\rm a}$ = 1.7 10$\\sp{10}$ M$\\sp{-1}$) complexed with fluorescyl ligand defined the six antibody active site contact residues involved in FI binding. For better definition of the relative role of each antigen contact residue in high affinity FI binding, each contact residue was changed to various amino acids in the single chain derivative of Mab 4-4-20 and following expression in Escherichia coli, denaturation, refolding and purification, each SCA (single chain antibody) mutant was characterized in terms of FI binding affinity, Q$\\sb{\\rm max}$ (maximum fluorescein fluorescence quenching), $\\lambda\\sb{\\rm max}$ (Absorption maxima) and idiotype. Alanine substitutions at the six ligand-contact residues reduced the SCA binding affinities and quenching maxima for all the Ala mutants except L27d which retained wild type binding characteristics. Results of Ala substitution mutagenesis suggested that L32$\\sp{\\rm Tyr}$, L91$\\sp{\\rm Ser}$ and H33$\\sp{\\rm Trp}$ are most important for high affinity FI binding and efficient FI quenching. Substitution of Tyr and Phe at residue H33 resulted in binding affinities that were greater than that obtained from the H33$\\sp{\\rm Ala}$ mutant and suggested that another aromatic amino acid could substitute for Trp this residue. A phenylalanine substitution at L32$\\sp{\\rm Tyr}$, which disrupted the Tyr hydrogen bond with fluorescyl ligand, resulted in a decreased the binding affinity ($\\sim$30-fold), but did not effect the quenching maxima. Finally, other amino acid substitutions at L34$\\sp{\\rm Arg}$, L91$\\sp{\\rm Ser}$ and L27d$\\sp{\\rm His}$ resulted in SCA mutants that possessed lower binding affinities and quenching maxima than that obtained for the respective Ala mutant.","Primary structure comparisons of idiotypically cross-reactive Mabs 4-4-20, 9-40, 12-40 and 5-14 possessed identical FI contact residues with the exception of L34His for L34Arg. Site specific mutagenesis studies of SCA 4-4-20 in which L34Arg was changed to L34His resulted in $\\sim$1000-fold and 3-fold decrease in binding affinity and Q$\\sb{\\rm max}$, respectively, which suggested that L34Arg was responsible for 4-4-20 increased binding affinity and fluorescence quenching. Collectively, these results suggest that the combining sites of Mab 9-40, 12-40 and 5-14 may possess different active site structures than Mab 4-4-20. (Abstract shortened by UMI.)","Made available in DSpace on 2011-05-07T12:57:23Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9305505.pdf: 5034851 bytes, checksum: 86b7fad0d4c99fb47f367ef3b6210718 (MD5) Previous issue date: 1992","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:48:15Z Item is restricted indefinitely."]},{"key":"dc:title","label":"Title","values":["The anti-fluorescein antibody active site: A single-chain site-specific mutagenesis study"]}]}],"canonical_facts":{"dc:contributor":["Voss, Edward W., Jr."],"dc:creator":["Denzin, Lisa Kae"],"dc:date":["2011-05-07T12:57:23Z","10000-01-01","1992"],"dc:description":["Restriction data tranferred 2014-07-01T11:21:50-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only","Crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (K$\\sb{\\rm a}$ = 1.7 10$\\sp{10}$ M$\\sp{-1}$) complexed with fluorescyl ligand defined the six antibody active site contact residues involved in FI binding. For better definition of the relative role of each antigen contact residue in high affinity FI binding, each contact residue was changed to various amino acids in the single chain derivative of Mab 4-4-20 and following expression in Escherichia coli, denaturation, refolding and purification, each SCA (single chain antibody) mutant was characterized in terms of FI binding affinity, Q$\\sb{\\rm max}$ (maximum fluorescein fluorescence quenching), $\\lambda\\sb{\\rm max}$ (Absorption maxima) and idiotype. Alanine substitutions at the six ligand-contact residues reduced the SCA binding affinities and quenching maxima for all the Ala mutants except L27d which retained wild type binding characteristics. Results of Ala substitution mutagenesis suggested that L32$\\sp{\\rm Tyr}$, L91$\\sp{\\rm Ser}$ and H33$\\sp{\\rm Trp}$ are most important for high affinity FI binding and efficient FI quenching. Substitution of Tyr and Phe at residue H33 resulted in binding affinities that were greater than that obtained from the H33$\\sp{\\rm Ala}$ mutant and suggested that another aromatic amino acid could substitute for Trp this residue. A phenylalanine substitution at L32$\\sp{\\rm Tyr}$, which disrupted the Tyr hydrogen bond with fluorescyl ligand, resulted in a decreased the binding affinity ($\\sim$30-fold), but did not effect the quenching maxima. Finally, other amino acid substitutions at L34$\\sp{\\rm Arg}$, L91$\\sp{\\rm Ser}$ and L27d$\\sp{\\rm His}$ resulted in SCA mutants that possessed lower binding affinities and quenching maxima than that obtained for the respective Ala mutant.","Primary structure comparisons of idiotypically cross-reactive Mabs 4-4-20, 9-40, 12-40 and 5-14 possessed identical FI contact residues with the exception of L34His for L34Arg. Site specific mutagenesis studies of SCA 4-4-20 in which L34Arg was changed to L34His resulted in $\\sim$1000-fold and 3-fold decrease in binding affinity and Q$\\sb{\\rm max}$, respectively, which suggested that L34Arg was responsible for 4-4-20 increased binding affinity and fluorescence quenching. Collectively, these results suggest that the combining sites of Mab 9-40, 12-40 and 5-14 may possess different active site structures than Mab 4-4-20. (Abstract shortened by UMI.)","Made available in DSpace on 2011-05-07T12:57:23Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9305505.pdf: 5034851 bytes, checksum: 86b7fad0d4c99fb47f367ef3b6210718 (MD5) Previous issue date: 1992","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:48:15Z Item is restricted indefinitely."],"dc:identifier":["AAI9305505","(UMI)AAI9305505","http://hdl.handle.net/2142/21067"],"dc:language":["eng"],"dc:rights":["Copyright 1992 Denzin, Lisa Kae"],"dc:subject":["Biology, Microbiology","Health Sciences, Immunology"],"dc:title":["The anti-fluorescein antibody active site: A single-chain site-specific mutagenesis study"],"dc:type":["text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:17Z"}