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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01kd17cw891
Title: Cryo-EM Structural Determination of Nav1.7-NaChBac Chimera and Df1a Toxin
Authors: On, Cam
Advisors: Yan, Nieng
Department: Molecular Biology
Class Year: 2020
Abstract: Ion regulation is an essential mechanism in the propagation of action potentials in nervous tissues. The influx of sodium ions is controlled by transmembrane Nav channels. Voltage-gated Nav1.7 is one such channel that propagates action potential in peripheral sensory neurons. Mutations in Nav1.7 have been linked to neurological disorders such as congenital indifference to pain, Dravet syndrome, and primary erythromelalgia. Furthermore, there are numerous animal toxins, such as Df1a, that act as gating modifiers to inhibit the channel, producing numbing effects. Therefore, Nav1.7 is a strong model for studying analgesics and channelopathies. Although the channel serves important medical and pharmacological purposes, decades of research have yet to completely elucidate the conformations and mechanisms utilized by Nav1.7 due to poor purification behavior. This investigation utilizes NaChBac, a prokaryotic equivalent to Nav1.7, to capture high resolution images and construct a model of the complex formed between Nav1.7 and Df1a through single-particle electron microscopy (Cryo-EM). The voltage-sensing domain II (VSDII) of Nav1.7, the site where Df1a is known to interface with, is substituted into NaChBac, producing a VSDII-NaChBac chimera. The chimera was reconstituted into nanodisc, a lipid bilayer construct, and vitrified for imaging. This study presents a 3.4 angstrom model of the chimera in nanodisc and a 3.7 angstrom model of the chimera and toxin complex in nanodisc. The results provide insight into protein purification, dynamics and imaging, useful for pharmacological framework and drug design in treating channelopathies. In addition, NaChBac proves to be a strong model for studying eukaryotic Nav channels. Further optimization of the study will elucidate the mechanisms of Df1a acting on Nav1.7.
URI: http://arks.princeton.edu/ark:/88435/dsp01kd17cw891
Type of Material: Princeton University Senior Theses
Language: en
Appears in Collections:Molecular Biology, 1954-2024

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