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The crystal structure of human Navβ3-Ig domain and its implications https://doi.org/10.17863/CAM.107561 Namadurai, Sivakumar Jackson, Antony Chirgadze, Dimitri The mammalian Voltage-gated sodium (Na_v) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Na_vβ subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Na_vα 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_vβ-subunit isoforms, Na_vβ1‒4 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_vβ3 subunit. It reports the atomic structure of the Na_vβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Na_vβ3-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_vβ3 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_vβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Na_vβ3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Na_vβ3, *in vivo*), Atomic Force Microscopy (isolated full-length Na_vβ3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Na_vβ3, *in situ*) support the view that the Na_vβ3 subunit can form trimers when expressed in cells. The biological significance of Na_vβ3 subunit trimerization is discussed. Strategies to express and purify the Na_vβ1/β2/β4-Ig domains were made. Wild type Na_vβ2- and Na_vβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced. University of Cambridge 2023-10-31 Thesis Doctoral Doctor of Philosophy (PhD) eng Cambridge Nehru Trust (partial) Scholarship St. John's College Bursary https://www.repository.cam.ac.uk/handle/1810/366790 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2be10d3e-b241-48fd-ab18-b555e5477a29/download 0d78d15bd68a7d5783c029066448c39d https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f57616d3-c0c4-4500-9442-893ca9b49d4d/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ crystal structure of Navβ3-Ig domain