Date of Degree

6-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Sebastien Poget

Committee Members

Sharon Loverde

Ming Tang

Reza Khayat

Crina Nimigean

Subject Categories

Physical Sciences and Mathematics

Keywords

Voltage Gated Sodium Channels

Abstract

Voltage gated sodium channels (VGSCs) are essential to the propagation of nerve cell impulses and thus communication in the nervous system. VGSCs are comprised of four repeating units of six transmembrane helices. Of the six transmembrane helices, the first four (called S1-S4) comprise the voltage sensing domain (VSD) and the last two (S5-S6) comprise the pore domain (PD)1. The Human voltage-gated sodium channel NaV1.7 has been shown to play a vital role in the nerve pathways that induce pain response2. Many of the currently available pain therapeutic drugs are opiates, derivative molecules of opium. These drugs target receptors in the brain that result in many other effects in addition to pain relief, such as euphoria, which gives such drugs a high risk for misuse. Drugs that target and inhibit NaV1.7 on the other hand should only affect the pain signal and therefore lead to less addiction problems than opiate-based medications. Animal peptide toxins with high specificity towards NaV1.7 can potentially serve as lead compounds for the design of such drugs. One example of such a toxin is the tarantula toxin Protoxin II (ProTxII). Although the cryo-EM structures of hNav1.7 in complex with ProTxII have been reported, the resolution of the toxin-channel binding interface is poor3 4. Therefore, obtaining a deeper understanding of the toxin-channel binding interface is still a crucial pursuit for the advancement of rational drug design in targeting NaV1.7 for potential pain relief. GpTxI is another tarantula toxin that has been shown to selectively bind to 5. GpTxI is of particular interest because the toxin itself has been shown to exhibit analgesic effects6. Therefore, it would be beneficial to obtain structural information on GpTxI-NaV1.7 interactions to help detect determinants of their relative specificity and binding affinity for the channel. In this study, we expressed, purified and refolded the voltage sensing domain (VSD) of repeat II of this channel with the aim of conducting ligand binding and structural studies via solution state NMR spectroscopy. We have successfully expressed the recombinant voltage sensing domain (VSD) by using the Trp-Delta-Ldr inclusion body system7. The fusion protein was cleaved with hydroxylamine using an added NG sequence between the fusion partners. The VSD was then refolded using the zwitterionic lipid, DMPC. Micro-scale thermophoresis (MST) was used to confirm binding of the recombinant VSD to Protoxin-II. Additionally, GpTxI was synthesized and refolded for binding and NMR studies with VSD-II. In this study, we show that VSD adopts a native-like conformation due the high-affinity binding between the VSD and ProTxII and GpTxI. Moreover, we have produced the mutations F813A and D816A in the S3/S4 linker region of the VSD and found the binding affinity towards GpTxI is significantly decreased. Thus, we have found residues of the VSD which are likely to be directly involved with the binding of Nav1.7 and the analgesic toxin, GpTxI. In addition, we have perofmed HSQC NMR experiments on the VSD in lyso-myristoyl phosphocholine (LMPC) micelles in aim to optimize NMR conditions for further investigation using 3-dimensional NMR experiments. In addition, we have solved the solution-state NMR structure of a novel Terebrid toxin from Terebra Subulata. The novel toxin, known as Tsu1.1, has the potential to target receptors that are involved with appetite signaling, as it has been found to significantly affect the apetite of Drosophila8. Also, being that this toxin has not been extensively investigated for targets, there could be numerous potential rational drug design applications in the structural findings of Tsu1.1.

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