{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/106447"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/106447","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Conformational engineering of human chemokine receptors and HIV-1 Env using deep mutational scanning","abstract":"Experimental analysis of protein mutations is classically a ‘small data’ problem. A limited number of mutants are made at sites of suspected importance, and the activity of each variant is individually tested. However, by combining directed evolution and deep sequencing with site- saturation mutagenesis (SSM) to create unbiased, diverse libraries, it is now possible to track the phenotypic fitness of thousands of sequence variants in a single experiment. This is known as deep mutational scanning, and it allows for the comprehensive experimental determination of a protein’s sequence-activity landscape. I applied deep mutational scanning to the human chemokine receptors CXCR4 and CCR5, which are well-studied co-receptors for HIV-1 infection. Libraries of both receptors containing nearly all ~7,000 single amino acid substitutions were selected in tissue culture for surface expression, binding to conformation-specific antibodies, binding to a chemokine, and binding to the HIV-1 surface glycoprotein Env. Using these landscapes, I identified that the chemokine CXCL12 binds asymmetrically in the CXCR4 ligand-binding cavity, discovered mutations within a potential allosteric site in CXCR4 that enhance CXCL12 binding, and engineered a CCR5 variant with increased Env binding for structural biology purposes. Building upon this method, I next deep mutationally scanned Env, the only HIV-1 protein exposed on the viral surface to the humoral immune system. Viral Env engages the host receptor CD4, triggering Env to transition from a ‘closed’ to ‘open’ conformation leading to virus-cell membrane fusion. To understand how HIV-1 Env sequence accommodates conformational changes, comprehensive mutational landscapes were determined for Env from the BaL and DU422 isolates interacting with CD4, or antibody PG16 that preferentially recognizes closed trimers. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this observation, mutations were found that increase presentation of quaternary epitopes associated with closed trimeric Env. Many of the mutations reduce electrostatic repulsion at the Env apex, or increase hydrophobic packing at the gp120 inner-outer domain interface. Furthermore, engineered Env variants containing core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. The suite of mutations was broadly applicable to Env sequences from HIV-1 or Simian-HIV strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. These findings may assist immunogen design. In summary, this body of work highlights a mutational scanning methodology that can be broadly applied to any complex protein expressed in human cells.","abstract_html":"Experimental analysis of protein mutations is classically a ‘small data’ problem. A limited number of mutants are made at sites of suspected importance, and the activity of each variant is individually tested. However, by combining directed evolution and deep sequencing with site- saturation mutagenesis (SSM) to create unbiased, diverse libraries, it is now possible to track the phenotypic fitness of thousands of sequence variants in a single experiment. This is known as deep mutational scanning, and it allows for the comprehensive experimental determination of a protein’s sequence-activity landscape. I applied deep mutational scanning to the human chemokine receptors CXCR4 and CCR5, which are well-studied co-receptors for HIV-1 infection. Libraries of both receptors containing nearly all ~7,000 single amino acid substitutions were selected in tissue culture for surface expression, binding to conformation-specific antibodies, binding to a chemokine, and binding to the HIV-1 surface glycoprotein Env. Using these landscapes, I identified that the chemokine CXCL12 binds asymmetrically in the CXCR4 ligand-binding cavity, discovered mutations within a potential allosteric site in CXCR4 that enhance CXCL12 binding, and engineered a CCR5 variant with increased Env binding for structural biology purposes. Building upon this method, I next deep mutationally scanned Env, the only HIV-1 protein exposed on the viral surface to the humoral immune system. Viral Env engages the host receptor CD4, triggering Env to transition from a ‘closed’ to ‘open’ conformation leading to virus-cell membrane fusion. To understand how HIV-1 Env sequence accommodates conformational changes, comprehensive mutational landscapes were determined for Env from the BaL and DU422 isolates interacting with CD4, or antibody PG16 that preferentially recognizes closed trimers. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this observation, mutations were found that increase presentation of quaternary epitopes associated with closed trimeric Env. Many of the mutations reduce electrostatic repulsion at the Env apex, or increase hydrophobic packing at the gp120 inner-outer domain interface. Furthermore, engineered Env variants containing core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. The suite of mutations was broadly applicable to Env sequences from HIV-1 or Simian-HIV strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. These findings may assist immunogen design. In summary, this body of work highlights a mutational scanning methodology that can be broadly applied to any complex protein expressed in human cells.","abstract_has_math":false,"creators":["Heredia, Jeremiah D."],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Procko, Erik","Brooke, Christopher","Kranz, David","Nair, Satish"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2020,"date_issued":"2020-03-02T22:38:44Z","date_published":"2020-03-02T22:38:44Z","updated_at":"2026-07-22T22:24:47Z","subjects":["deep mutational scan","directed evolution","GPCRs","mammalian surface display","protein engineering","protein dynamics","gp160","human immunodeficiency virus","quaternary epitope","CD4","Env","broadly neutralizing antibody","conformational change","CCR5","CXCR4","chemokine receptors","sweet taste receptors","T1R2","T1R3","SERT","genetics and disease"],"languages":["en"],"rights":["Copyright 2019 Jeremiah Heredia"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/106447","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Procko, Erik","Brooke, Christopher","Kranz, David","Nair, Satish"]},{"key":"dc:creator","label":"Author","values":["Heredia, Jeremiah D."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2020-03-02T22:38:44Z","2022-03-03T10:15:13Z","2019-11-19","2019-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"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":["deep mutational scan","directed evolution","GPCRs","mammalian surface display","protein engineering","protein dynamics","gp160","human immunodeficiency virus","quaternary epitope","CD4","Env","broadly neutralizing antibody","conformational change","CCR5","CXCR4","chemokine receptors","sweet taste receptors","T1R2","T1R3","SERT","genetics and disease"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2019 Jeremiah Heredia"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/106447"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Experimental analysis of protein mutations is classically a ‘small data’ problem. A limited number of mutants are made at sites of suspected importance, and the activity of each variant is individually tested. However, by combining directed evolution and deep sequencing with site- saturation mutagenesis (SSM) to create unbiased, diverse libraries, it is now possible to track the phenotypic fitness of thousands of sequence variants in a single experiment. This is known as deep mutational scanning, and it allows for the comprehensive experimental determination of a protein’s sequence-activity landscape. I applied deep mutational scanning to the human chemokine receptors CXCR4 and CCR5, which are well-studied co-receptors for HIV-1 infection. Libraries of both receptors containing nearly all ~7,000 single amino acid substitutions were selected in tissue culture for surface expression, binding to conformation-specific antibodies, binding to a chemokine, and binding to the HIV-1 surface glycoprotein Env. Using these landscapes, I identified that the chemokine CXCL12 binds asymmetrically in the CXCR4 ligand-binding cavity, discovered mutations within a potential allosteric site in CXCR4 that enhance CXCL12 binding, and engineered a CCR5 variant with increased Env binding for structural biology purposes. Building upon this method, I next deep mutationally scanned Env, the only HIV-1 protein exposed on the viral surface to the humoral immune system. Viral Env engages the host receptor CD4, triggering Env to transition from a ‘closed’ to ‘open’ conformation leading to virus-cell membrane fusion. To understand how HIV-1 Env sequence accommodates conformational changes, comprehensive mutational landscapes were determined for Env from the BaL and DU422 isolates interacting with CD4, or antibody PG16 that preferentially recognizes closed trimers. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this observation, mutations were found that increase presentation of quaternary epitopes associated with closed trimeric Env. Many of the mutations reduce electrostatic repulsion at the Env apex, or increase hydrophobic packing at the gp120 inner-outer domain interface. Furthermore, engineered Env variants containing core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. The suite of mutations was broadly applicable to Env sequences from HIV-1 or Simian-HIV strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. These findings may assist immunogen design. In summary, this body of work highlights a mutational scanning methodology that can be broadly applied to any complex protein expressed in human cells.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2021-12-01","The student, Jeremiah Heredia, accepted the attached license on 2019-11-18 at 10:54.","The student, Jeremiah Heredia, submitted this Dissertation for approval on 2019-11-18 at 11:15.","This Dissertation was approved for publication on 2019-11-19 at 09:21.","DSpace SAF Submission Ingestion Package generated from Vireo submission #14560 on 2020-02-28 at 17:36:22","Made available in DSpace on 2020-03-02T22:38:44Z (GMT). No. of bitstreams: 2 HEREDIA-DISSERTATION-2019.pdf: 18942225 bytes, checksum: 1d304bb3db4171aa8f64914d64c4ac9d (MD5) LICENSE.txt: 4213 bytes, checksum: 9585b7ca79358822b66602578519fdf9 (MD5) Previous issue date: 2019-11-19","Embargo set by: Seth Robbins for item 113991 Lift date: 2022-03-02T22:39:04Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 113991 on 2022-03-03T10:15:13Z."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Conformational engineering of human chemokine receptors and HIV-1 Env using deep mutational scanning"]}]}],"canonical_facts":{"dc:contributor":["Procko, Erik","Brooke, Christopher","Kranz, David","Nair, Satish"],"dc:creator":["Heredia, Jeremiah D."],"dc:date":["2020-03-02T22:38:44Z","2022-03-03T10:15:13Z","2019-11-19","2019-12"],"dc:description":["Experimental analysis of protein mutations is classically a ‘small data’ problem. A limited number of mutants are made at sites of suspected importance, and the activity of each variant is individually tested. However, by combining directed evolution and deep sequencing with site- saturation mutagenesis (SSM) to create unbiased, diverse libraries, it is now possible to track the phenotypic fitness of thousands of sequence variants in a single experiment. This is known as deep mutational scanning, and it allows for the comprehensive experimental determination of a protein’s sequence-activity landscape. I applied deep mutational scanning to the human chemokine receptors CXCR4 and CCR5, which are well-studied co-receptors for HIV-1 infection. Libraries of both receptors containing nearly all ~7,000 single amino acid substitutions were selected in tissue culture for surface expression, binding to conformation-specific antibodies, binding to a chemokine, and binding to the HIV-1 surface glycoprotein Env. Using these landscapes, I identified that the chemokine CXCL12 binds asymmetrically in the CXCR4 ligand-binding cavity, discovered mutations within a potential allosteric site in CXCR4 that enhance CXCL12 binding, and engineered a CCR5 variant with increased Env binding for structural biology purposes. Building upon this method, I next deep mutationally scanned Env, the only HIV-1 protein exposed on the viral surface to the humoral immune system. Viral Env engages the host receptor CD4, triggering Env to transition from a ‘closed’ to ‘open’ conformation leading to virus-cell membrane fusion. To understand how HIV-1 Env sequence accommodates conformational changes, comprehensive mutational landscapes were determined for Env from the BaL and DU422 isolates interacting with CD4, or antibody PG16 that preferentially recognizes closed trimers. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this observation, mutations were found that increase presentation of quaternary epitopes associated with closed trimeric Env. Many of the mutations reduce electrostatic repulsion at the Env apex, or increase hydrophobic packing at the gp120 inner-outer domain interface. Furthermore, engineered Env variants containing core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. The suite of mutations was broadly applicable to Env sequences from HIV-1 or Simian-HIV strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. These findings may assist immunogen design. In summary, this body of work highlights a mutational scanning methodology that can be broadly applied to any complex protein expressed in human cells.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2021-12-01","The student, Jeremiah Heredia, accepted the attached license on 2019-11-18 at 10:54.","The student, Jeremiah Heredia, submitted this Dissertation for approval on 2019-11-18 at 11:15.","This Dissertation was approved for publication on 2019-11-19 at 09:21.","DSpace SAF Submission Ingestion Package generated from Vireo submission #14560 on 2020-02-28 at 17:36:22","Made available in DSpace on 2020-03-02T22:38:44Z (GMT). No. of bitstreams: 2 HEREDIA-DISSERTATION-2019.pdf: 18942225 bytes, checksum: 1d304bb3db4171aa8f64914d64c4ac9d (MD5) LICENSE.txt: 4213 bytes, checksum: 9585b7ca79358822b66602578519fdf9 (MD5) Previous issue date: 2019-11-19","Embargo set by: Seth Robbins for item 113991 Lift date: 2022-03-02T22:39:04Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 113991 on 2022-03-03T10:15:13Z."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/106447"],"dc:language":["en"],"dc:rights":["Copyright 2019 Jeremiah Heredia"],"dc:subject":["deep mutational scan","directed evolution","GPCRs","mammalian surface display","protein engineering","protein dynamics","gp160","human immunodeficiency virus","quaternary epitope","CD4","Env","broadly neutralizing antibody","conformational change","CCR5","CXCR4","chemokine receptors","sweet taste receptors","T1R2","T1R3","SERT","genetics and disease"],"dc:title":["Conformational engineering of human chemokine receptors and HIV-1 Env using deep mutational scanning"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:47Z"}