{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/22853"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/22853","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis","abstract":"Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I.","abstract_html":"Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I.","abstract_has_math":true,"creators":["Ji, Yong"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Jonas, Ana"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T13:53:40Z","date_published":"2011-05-07T13:53:40Z","updated_at":"2026-07-22T22:25:20Z","subjects":["Biology, Molecular","Chemistry, Biochemistry","Health Sciences, General"],"languages":["eng"],"rights":["Copyright 1996 Ji, Yong"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["9780591087949","AAI9702551","(UMI)AAI9702551"],"render_values":[{"text":"9780591087949","href":null,"code":true},{"text":"AAI9702551","href":null,"code":true},{"text":"(UMI)AAI9702551","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/22853","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Jonas, Ana"]},{"key":"dc:creator","label":"Author","values":["Ji, Yong"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T13:53:40Z","10000-01-01","1996"]},{"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":["Biology, Molecular","Chemistry, Biochemistry","Health Sciences, General"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1996 Ji, Yong"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["9780591087949","AAI9702551","(UMI)AAI9702551","http://hdl.handle.net/2142/22853"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I.","In aqueous solution the fragment, apoA-I(1-192), had a similar $\\alpha$-helical content to apoA-I ($\\sim$52%) but existed only as monomers and dimers. ApoA-I(1-192) lysed dimyristoyl PC (DMPC) liposomes slowly compared with apoA-I, but did form rHDL complexes with POPC or DPPC when prepared by the sodium cholate dialysis method. The isolated apoA-I(1-192) rHDLs activated lecithin cholesterol acyltransferase (LCAT), promoted cholesterol efflux from rat heptoma cells, and bound tightly to HeLa cells, indicating that the C-terminal 51 residues of apoA-I are crucial for self-association and initial lipid-binding but are not required for specific LCAT activation, cholesterol efflux or cell surface binding.","A highly efficient system was developed for overexpressing mature apoA-I and two C-terminal deletion mutants, apoA-I(1-164) and apoA-I(1-186), in E. coli. The recombinant apoA-I constituted more than 15% of the total cell protein. ApoA-I(1-164) was produced to a much lower level ($\\sim$2-fold) than the other two proteins, suggesting that the C-terminal domain of apoA-I protects the protein from degradation. Essentially pure ($>$95%) apoA-I(1-164) and apoA-I(1-186) were obtained by reverse phase HPLC, giving a final yield for apoA-I(1-164) of $\\sim$2 mg/liter culture.","ApoA-I(1-164) formed more dimers than monomers and had a lower $\\alpha$-helical content than apoA-I(1-192); its Trp residues were more exposed to the aqueous environment. ApoA-I(1-164) exhibited decreased lipid-binding ability, indicating that the region between 165 and 192 is involved in lipid-protein interactions. Nonetheless, apoA-I(1-164) bound lipids cooperatively and formed POPC rHDL of distinct and defined sizes when prepared by the sodium cholate method.","Made available in DSpace on 2011-05-07T13:53:40Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9702551.pdf: 4935507 bytes, checksum: 9ae3315318d73864281184ba050b4216 (MD5) Previous issue date: 1996","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T15:00:28Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:28:36-05:00 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"]},{"key":"dc:title","label":"Title","values":["Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis"]}]}],"canonical_facts":{"dc:contributor":["Jonas, Ana"],"dc:creator":["Ji, Yong"],"dc:date":["2011-05-07T13:53:40Z","10000-01-01","1996"],"dc:description":["Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I.","In aqueous solution the fragment, apoA-I(1-192), had a similar $\\alpha$-helical content to apoA-I ($\\sim$52%) but existed only as monomers and dimers. ApoA-I(1-192) lysed dimyristoyl PC (DMPC) liposomes slowly compared with apoA-I, but did form rHDL complexes with POPC or DPPC when prepared by the sodium cholate dialysis method. The isolated apoA-I(1-192) rHDLs activated lecithin cholesterol acyltransferase (LCAT), promoted cholesterol efflux from rat heptoma cells, and bound tightly to HeLa cells, indicating that the C-terminal 51 residues of apoA-I are crucial for self-association and initial lipid-binding but are not required for specific LCAT activation, cholesterol efflux or cell surface binding.","A highly efficient system was developed for overexpressing mature apoA-I and two C-terminal deletion mutants, apoA-I(1-164) and apoA-I(1-186), in E. coli. The recombinant apoA-I constituted more than 15% of the total cell protein. ApoA-I(1-164) was produced to a much lower level ($\\sim$2-fold) than the other two proteins, suggesting that the C-terminal domain of apoA-I protects the protein from degradation. Essentially pure ($>$95%) apoA-I(1-164) and apoA-I(1-186) were obtained by reverse phase HPLC, giving a final yield for apoA-I(1-164) of $\\sim$2 mg/liter culture.","ApoA-I(1-164) formed more dimers than monomers and had a lower $\\alpha$-helical content than apoA-I(1-192); its Trp residues were more exposed to the aqueous environment. ApoA-I(1-164) exhibited decreased lipid-binding ability, indicating that the region between 165 and 192 is involved in lipid-protein interactions. Nonetheless, apoA-I(1-164) bound lipids cooperatively and formed POPC rHDL of distinct and defined sizes when prepared by the sodium cholate method.","Made available in DSpace on 2011-05-07T13:53:40Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9702551.pdf: 4935507 bytes, checksum: 9ae3315318d73864281184ba050b4216 (MD5) Previous issue date: 1996","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T15:00:28Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:28:36-05:00 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"],"dc:identifier":["9780591087949","AAI9702551","(UMI)AAI9702551","http://hdl.handle.net/2142/22853"],"dc:language":["eng"],"dc:rights":["Copyright 1996 Ji, Yong"],"dc:subject":["Biology, Molecular","Chemistry, Biochemistry","Health Sciences, General"],"dc:title":["Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis"],"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":"2026-07-22T22:25:20Z"}