{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/366790"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/366790","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"The crystal structure of human Navβ3-Ig domain and its implications","abstract":"The mammalian Voltage-gated sodium (Na<sub>v</sub>) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na<sub>v</sub>β subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na<sub>v</sub>α subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Na<sub>v</sub>β-subunit isoforms, Na<sub>v</sub>β1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Na<sub>v</sub>β3 subunit. It reports the atomic structure of the Na<sub>v</sub>β3-Ig domain as determined by X-ray crystallography. Interestingly, the Na<sub>v</sub>β3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Na<sub>v</sub>β3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Na<sub>v</sub>β3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na<sub>v</sub>β3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na<sub>v</sub>β3, *in vivo*), Atomic Force Microscopy (isolated full-length Na<sub>v</sub>β3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na<sub>v</sub>β3, *in situ*) support the view that the Na<sub>v</sub>β3 subunit can form trimers when expressed in cells. The biological significance of Na<sub>v</sub>β3 subunit trimerization is discussed. Strategies to express and purify the Na<sub>v</sub>β1/β2/β4-Ig domains were made. Wild type Na<sub>v</sub>β2- and Na<sub>v</sub>β4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced.","abstract_html":"The mammalian Voltage-gated sodium (Na&lt;sub&gt;v&lt;/sub&gt;) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na&lt;sub&gt;v&lt;/sub&gt;β subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na&lt;sub&gt;v&lt;/sub&gt;α subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Na&lt;sub&gt;v&lt;/sub&gt;β-subunit isoforms, Na&lt;sub&gt;v&lt;/sub&gt;β1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Na&lt;sub&gt;v&lt;/sub&gt;β3 subunit. It reports the atomic structure of the Na&lt;sub&gt;v&lt;/sub&gt;β3-Ig domain as determined by X-ray crystallography. Interestingly, the Na&lt;sub&gt;v&lt;/sub&gt;β3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Na&lt;sub&gt;v&lt;/sub&gt;β3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Na&lt;sub&gt;v&lt;/sub&gt;β3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na&lt;sub&gt;v&lt;/sub&gt;β3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na&lt;sub&gt;v&lt;/sub&gt;β3, *in vivo*), Atomic Force Microscopy (isolated full-length Na&lt;sub&gt;v&lt;/sub&gt;β3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na&lt;sub&gt;v&lt;/sub&gt;β3, *in situ*) support the view that the Na&lt;sub&gt;v&lt;/sub&gt;β3 subunit can form trimers when expressed in cells. The biological significance of Na&lt;sub&gt;v&lt;/sub&gt;β3 subunit trimerization is discussed. Strategies to express and purify the Na&lt;sub&gt;v&lt;/sub&gt;β1/β2/β4-Ig domains were made. Wild type Na&lt;sub&gt;v&lt;/sub&gt;β2- and Na&lt;sub&gt;v&lt;/sub&gt;β4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced.","abstract_has_math":false,"creators":["Namadurai, Sivakumar"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Jackson, Antony","Chirgadze, Dimitri"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-10-31","date_published":"2023-10-31","updated_at":"2026-07-22T22:23:59Z","subjects":["crystal structure of Navβ3-Ig domain"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f57616d3-c0c4-4500-9442-893ca9b49d4d/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.107561","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Jackson, Antony","Chirgadze, Dimitri"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Cambridge Nehru Trust (partial) Scholarship St. John's College Bursary"]},{"key":"dc:creator","label":"Author","values":["Namadurai, Sivakumar"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-10-31"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/366790"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["crystal structure of Navβ3-Ig domain"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f57616d3-c0c4-4500-9442-893ca9b49d4d/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.107561"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2be10d3e-b241-48fd-ab18-b555e5477a29/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The mammalian Voltage-gated sodium (Na<sub>v</sub>) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na<sub>v</sub>β subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na<sub>v</sub>α subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Na<sub>v</sub>β-subunit isoforms, Na<sub>v</sub>β1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Na<sub>v</sub>β3 subunit. It reports the atomic structure of the Na<sub>v</sub>β3-Ig domain as determined by X-ray crystallography. Interestingly, the Na<sub>v</sub>β3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Na<sub>v</sub>β3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Na<sub>v</sub>β3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na<sub>v</sub>β3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na<sub>v</sub>β3, *in vivo*), Atomic Force Microscopy (isolated full-length Na<sub>v</sub>β3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na<sub>v</sub>β3, *in situ*) support the view that the Na<sub>v</sub>β3 subunit can form trimers when expressed in cells. The biological significance of Na<sub>v</sub>β3 subunit trimerization is discussed. Strategies to express and purify the Na<sub>v</sub>β1/β2/β4-Ig domains were made. Wild type Na<sub>v</sub>β2- and Na<sub>v</sub>β4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["0d78d15bd68a7d5783c029066448c39d","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["The crystal structure of human Navβ3-Ig domain and its implications"]}]}],"canonical_facts":{"dc:contributor.advisor":["Jackson, Antony","Chirgadze, Dimitri"],"dc:contributor.sponsor":["Cambridge Nehru Trust (partial) Scholarship St. John's College Bursary"],"dc:creator":["Namadurai, Sivakumar"],"dc:date.issued":["2023-10-31"],"dc:description.abstract":["The mammalian Voltage-gated sodium (Na<sub>v</sub>) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na<sub>v</sub>β subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na<sub>v</sub>α subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Na<sub>v</sub>β-subunit isoforms, Na<sub>v</sub>β1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Na<sub>v</sub>β3 subunit. It reports the atomic structure of the Na<sub>v</sub>β3-Ig domain as determined by X-ray crystallography. Interestingly, the Na<sub>v</sub>β3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Na<sub>v</sub>β3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Na<sub>v</sub>β3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na<sub>v</sub>β3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na<sub>v</sub>β3, *in vivo*), Atomic Force Microscopy (isolated full-length Na<sub>v</sub>β3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na<sub>v</sub>β3, *in situ*) support the view that the Na<sub>v</sub>β3 subunit can form trimers when expressed in cells. The biological significance of Na<sub>v</sub>β3 subunit trimerization is discussed. Strategies to express and purify the Na<sub>v</sub>β1/β2/β4-Ig domains were made. Wild type Na<sub>v</sub>β2- and Na<sub>v</sub>β4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced."],"dc:format.checksum.md5":["0d78d15bd68a7d5783c029066448c39d","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.107561"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2be10d3e-b241-48fd-ab18-b555e5477a29/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/366790"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f57616d3-c0c4-4500-9442-893ca9b49d4d/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["crystal structure of Navβ3-Ig domain"],"dc:title":["The crystal structure of human Navβ3-Ig domain and its implications"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:59Z"}