{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/88142"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/88142","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Towards atomistic characterization of blood coagulation proteins by NMR spectroscopy","abstract":"Proteins involved in the blood coagulation cascade are of vital biomedical importance, and most essential blood-clotting reactions occur preferentially on phospholipid membranes. Understanding the interactions between the proteins and membranes involved in blood clotting is crucial to the continued development of successful treatments for blood coagulation disorders such as hemophilia, von Willebrand disease, and thrombosis. In normal hemostasis, the blood-clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to human tissue factor (TF), a 29 kDa integral membrane protein. The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the extracellular domain of TF (sTF) exist; however, some of these structures are lacking electron density in functionally important dynamic loops that can be accessed using nuclear magnetic resonance (NMR) spectroscopy. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR and compared to the previously published solution NMR sTF assignments. We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes, precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional solid-state NMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data are presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF in atomistic detail. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR (SSNMR) and compared to the previously published solution NMR sTF assignments (1). We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes (2), precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional SSNMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data will be presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies will provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF at atomistic detail.","abstract_html":"Proteins involved in the blood coagulation cascade are of vital biomedical importance, and most essential blood-clotting reactions occur preferentially on phospholipid membranes. Understanding the interactions between the proteins and membranes involved in blood clotting is crucial to the continued development of successful treatments for blood coagulation disorders such as hemophilia, von Willebrand disease, and thrombosis. In normal hemostasis, the blood-clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to human tissue factor (TF), a 29 kDa integral membrane protein. The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the extracellular domain of TF (sTF) exist; however, some of these structures are lacking electron density in functionally important dynamic loops that can be accessed using nuclear magnetic resonance (NMR) spectroscopy. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR and compared to the previously published solution NMR sTF assignments. We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes, precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional solid-state NMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data are presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF in atomistic detail. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR (SSNMR) and compared to the previously published solution NMR sTF assignments (1). We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes (2), precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional SSNMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data will be presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies will provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF at atomistic detail.","abstract_has_math":false,"creators":["Nuzzio, Kristin Marie"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Rienstra, Chad M.","Morrissey, James H.","Tajkhorshid, Emad","Gruebele, Martin"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-29T20:49:29Z","date_published":"2015-09-29T20:49:29Z","updated_at":"2026-07-22T22:26:31Z","subjects":["blood coagulation proteins","tissue factor","soluble tissue factor","Nuclear Magnetic Resonance (NMR) spectroscopy","solution nuclear magnetic resonance (NMR)","solid-state nuclear magnetic resonance (NMR)","protein expression","protein purification","chemical shift assignment","protein dynamics"],"languages":["en"],"rights":["Copyright 2015 Kristin M. Nuzzio"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/88142","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Rienstra, Chad M.","Morrissey, James H.","Tajkhorshid, Emad","Gruebele, Martin"]},{"key":"dc:creator","label":"Author","values":["Nuzzio, Kristin Marie"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-29T20:49:29Z","2017-09-30T09:15:35Z","2015-08","2015-06-15","2015-8"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"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":["blood coagulation proteins","tissue factor","soluble tissue factor","Nuclear Magnetic Resonance (NMR) spectroscopy","solution nuclear magnetic resonance (NMR)","solid-state nuclear magnetic resonance (NMR)","protein expression","protein purification","chemical shift assignment","protein dynamics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2015 Kristin M. Nuzzio"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/88142"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Proteins involved in the blood coagulation cascade are of vital biomedical importance, and most essential blood-clotting reactions occur preferentially on phospholipid membranes. Understanding the interactions between the proteins and membranes involved in blood clotting is crucial to the continued development of successful treatments for blood coagulation disorders such as hemophilia, von Willebrand disease, and thrombosis. In normal hemostasis, the blood-clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to human tissue factor (TF), a 29 kDa integral membrane protein. The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the extracellular domain of TF (sTF) exist; however, some of these structures are lacking electron density in functionally important dynamic loops that can be accessed using nuclear magnetic resonance (NMR) spectroscopy. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR and compared to the previously published solution NMR sTF assignments. We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes, precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional solid-state NMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data are presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF in atomistic detail. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR (SSNMR) and compared to the previously published solution NMR sTF assignments (1). We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes (2), precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional SSNMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data will be presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies will provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF at atomistic detail.","Submission published under a 24 month embargo labeled 'U of I only', the embargo will last until 2017-08-01","The student, Kristin Nuzzio, accepted the attached license on 2015-06-09 at 16:03.","The student, Kristin Nuzzio, submitted this Dissertation for approval on 2015-06-09 at 16:15.","This Dissertation was approved for publication on 2015-06-15 at 08:41.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8271 on 2015-09-29 at 14:58:31","Made available in DSpace on 2015-09-29T20:49:29Z (GMT). No. of bitstreams: 2 NUZZIO-DISSERTATION-2015.pdf: 30440680 bytes, checksum: 0b29744a60355dd23ffce991589af226 (MD5) LICENSE.txt: 4211 bytes, checksum: 37c3ab86012dac0b7a90c09a32e1294b (MD5) Previous issue date: 2015-06-15","Embargo set by: Seth Robbins for item 89422 Lift date: 2017-09-29T20:50:34Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only Restriction Lifted for Item 89422 on 2017-09-30T09:15:35Z."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Towards atomistic characterization of blood coagulation proteins by NMR spectroscopy"]}]}],"canonical_facts":{"dc:contributor":["Rienstra, Chad M.","Morrissey, James H.","Tajkhorshid, Emad","Gruebele, Martin"],"dc:creator":["Nuzzio, Kristin Marie"],"dc:date":["2015-09-29T20:49:29Z","2017-09-30T09:15:35Z","2015-08","2015-06-15","2015-8"],"dc:description":["Proteins involved in the blood coagulation cascade are of vital biomedical importance, and most essential blood-clotting reactions occur preferentially on phospholipid membranes. Understanding the interactions between the proteins and membranes involved in blood clotting is crucial to the continued development of successful treatments for blood coagulation disorders such as hemophilia, von Willebrand disease, and thrombosis. In normal hemostasis, the blood-clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to human tissue factor (TF), a 29 kDa integral membrane protein. The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the extracellular domain of TF (sTF) exist; however, some of these structures are lacking electron density in functionally important dynamic loops that can be accessed using nuclear magnetic resonance (NMR) spectroscopy. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR and compared to the previously published solution NMR sTF assignments. We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes, precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional solid-state NMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data are presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF in atomistic detail. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR (SSNMR) and compared to the previously published solution NMR sTF assignments (1). We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes (2), precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional SSNMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data will be presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies will provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF at atomistic detail.","Submission published under a 24 month embargo labeled 'U of I only', the embargo will last until 2017-08-01","The student, Kristin Nuzzio, accepted the attached license on 2015-06-09 at 16:03.","The student, Kristin Nuzzio, submitted this Dissertation for approval on 2015-06-09 at 16:15.","This Dissertation was approved for publication on 2015-06-15 at 08:41.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8271 on 2015-09-29 at 14:58:31","Made available in DSpace on 2015-09-29T20:49:29Z (GMT). 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