{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/101364"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/101364","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Engineering the interface of cytochrome b5 and myoglobin for in vitro and in vivo applications","abstract":"Heme-copper oxidases (HCOs) are large membrane proteins found in both bacteria and in eukaryotes. They catalyze about 90% of oxygen reduction in the atmosphere. They utilize a heme-copper center, which is comprised of a heme and a copper ion coordinated with three histidines, to catalyze the four-electron reduction of oxygen to water. Unfortunately, native enzymes are difficult to study because they are difficult to purify, and the presence of multiple metal cofactors make it difficult to characterize. Therefore, there is a need for small protein models that are able to mimic the native systems. Our lab was able to incorporate the heme-copper center found in HCOs in a smaller scaffold, Myoglobin, and the resulting mutant was named CuBMb. When our protein model was paired with its physiological electron transfer partner, cytochrome b5, fast electron transfer and O2 consumption was achieved, making our protein model system to have a catalytic rate similar to that of native HCOs. A key to the above success was engineering the electrostatic interactions between CuBMb and cytochrome b5. As a result, the system works well only when the ionic strength of the buffer is <5 mM, which is outside the common physiological ionic strength. As a result, applications for this system are limited. To overcome this limitation, cytochrome b5 was engineered such that the interaction with myoglobin became hydrophobic, which allowed for a broader application scope. The mutants were designed using Rosetta, and their oxidase activity was characterized. However, after two rounds of Rosetta, the designed mutants had lower oxidase activity compared to the native at any ionic strength.","abstract_html":"Heme-copper oxidases (HCOs) are large membrane proteins found in both bacteria and in eukaryotes. They catalyze about 90% of oxygen reduction in the atmosphere. They utilize a heme-copper center, which is comprised of a heme and a copper ion coordinated with three histidines, to catalyze the four-electron reduction of oxygen to water. Unfortunately, native enzymes are difficult to study because they are difficult to purify, and the presence of multiple metal cofactors make it difficult to characterize. Therefore, there is a need for small protein models that are able to mimic the native systems. Our lab was able to incorporate the heme-copper center found in HCOs in a smaller scaffold, Myoglobin, and the resulting mutant was named CuBMb. When our protein model was paired with its physiological electron transfer partner, cytochrome b5, fast electron transfer and O2 consumption was achieved, making our protein model system to have a catalytic rate similar to that of native HCOs. A key to the above success was engineering the electrostatic interactions between CuBMb and cytochrome b5. As a result, the system works well only when the ionic strength of the buffer is &lt;5 mM, which is outside the common physiological ionic strength. As a result, applications for this system are limited. To overcome this limitation, cytochrome b5 was engineered such that the interaction with myoglobin became hydrophobic, which allowed for a broader application scope. The mutants were designed using Rosetta, and their oxidase activity was characterized. However, after two rounds of Rosetta, the designed mutants had lower oxidase activity compared to the native at any ionic strength.","abstract_has_math":false,"creators":["Chan, Tracy Ying"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"M.S.","degree_level":"Thesis","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Lu, Yi"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-09-04T20:47:28Z","date_published":"2018-09-04T20:47:28Z","updated_at":"2026-07-22T22:24:38Z","subjects":["metalloproteins","protein-protein interfaces"],"languages":["en"],"rights":["Copyright 2018 Tracy Chan"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/101364","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Lu, Yi"]},{"key":"dc:creator","label":"Author","values":["Chan, Tracy Ying"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-09-04T20:47:28Z","2020-09-05T09:15:20Z","2018-04-23","2018-05"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M.S."]},{"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":["metalloproteins","protein-protein interfaces"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2018 Tracy Chan"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/101364"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Heme-copper oxidases (HCOs) are large membrane proteins found in both bacteria and in eukaryotes. They catalyze about 90% of oxygen reduction in the atmosphere. They utilize a heme-copper center, which is comprised of a heme and a copper ion coordinated with three histidines, to catalyze the four-electron reduction of oxygen to water. Unfortunately, native enzymes are difficult to study because they are difficult to purify, and the presence of multiple metal cofactors make it difficult to characterize. Therefore, there is a need for small protein models that are able to mimic the native systems. Our lab was able to incorporate the heme-copper center found in HCOs in a smaller scaffold, Myoglobin, and the resulting mutant was named CuBMb. When our protein model was paired with its physiological electron transfer partner, cytochrome b5, fast electron transfer and O2 consumption was achieved, making our protein model system to have a catalytic rate similar to that of native HCOs. A key to the above success was engineering the electrostatic interactions between CuBMb and cytochrome b5. As a result, the system works well only when the ionic strength of the buffer is <5 mM, which is outside the common physiological ionic strength. As a result, applications for this system are limited. To overcome this limitation, cytochrome b5 was engineered such that the interaction with myoglobin became hydrophobic, which allowed for a broader application scope. The mutants were designed using Rosetta, and their oxidase activity was characterized. However, after two rounds of Rosetta, the designed mutants had lower oxidase activity compared to the native at any ionic strength.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-05-01","The student, Tracy Chan, accepted the attached license on 2018-04-20 at 16:16.","The student, Tracy Chan, submitted this Thesis for approval on 2018-04-20 at 16:23.","This Thesis was approved for publication on 2018-04-23 at 09:51.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12409 on 2018-08-31 at 17:30:14","Made available in DSpace on 2018-09-04T20:47:28Z (GMT). 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They catalyze about 90% of oxygen reduction in the atmosphere. They utilize a heme-copper center, which is comprised of a heme and a copper ion coordinated with three histidines, to catalyze the four-electron reduction of oxygen to water. Unfortunately, native enzymes are difficult to study because they are difficult to purify, and the presence of multiple metal cofactors make it difficult to characterize. Therefore, there is a need for small protein models that are able to mimic the native systems. Our lab was able to incorporate the heme-copper center found in HCOs in a smaller scaffold, Myoglobin, and the resulting mutant was named CuBMb. When our protein model was paired with its physiological electron transfer partner, cytochrome b5, fast electron transfer and O2 consumption was achieved, making our protein model system to have a catalytic rate similar to that of native HCOs. A key to the above success was engineering the electrostatic interactions between CuBMb and cytochrome b5. As a result, the system works well only when the ionic strength of the buffer is <5 mM, which is outside the common physiological ionic strength. As a result, applications for this system are limited. To overcome this limitation, cytochrome b5 was engineered such that the interaction with myoglobin became hydrophobic, which allowed for a broader application scope. The mutants were designed using Rosetta, and their oxidase activity was characterized. However, after two rounds of Rosetta, the designed mutants had lower oxidase activity compared to the native at any ionic strength.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-05-01","The student, Tracy Chan, accepted the attached license on 2018-04-20 at 16:16.","The student, Tracy Chan, submitted this Thesis for approval on 2018-04-20 at 16:23.","This Thesis was approved for publication on 2018-04-23 at 09:51.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12409 on 2018-08-31 at 17:30:14","Made available in DSpace on 2018-09-04T20:47:28Z (GMT). No. of bitstreams: 2 CHAN-THESIS-2018.pdf: 1607534 bytes, checksum: acb89d80898641a44c18aaa44d9ad2ec (MD5) LICENSE.txt: 4207 bytes, checksum: e40029aaae15242d51c0b9501d0b0419 (MD5) Previous issue date: 2018-04-23","Embargo set by: Seth Robbins for item 107449 Lift date: 2020-09-04T20:47:38Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 107449 Lift date: 2020-09-04T20:50:11Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 107449 on 2020-09-05T09:15:20Z."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/101364"],"dc:language":["en"],"dc:rights":["Copyright 2018 Tracy Chan"],"dc:subject":["metalloproteins","protein-protein interfaces"],"dc:title":["Engineering the interface of cytochrome b5 and myoglobin for in vitro and in vivo applications"],"dc:type":["text"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["M.S."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:38Z"}