Authors: Nys, Mieke
Issue Date: 23-May-2016
Abstract: The research in the lab of structural neurobiology is focused on the structure-function relationship of ion channels. Ion channels are dynamic proteins embedded in the lipid rich cell membrane. They can be activated by a wide range of stimuli, including changes in membrane potential, a temperature decrease or increase, mechanical inputs and the binding of ligands. The binding of neurotransmitters to pentameric ligand-gated ion channels (pLGICs), for example, results in the opening of these channels so that they become permeable for ions. pLGICs are mainly located in the peripheral and central nervous system, where they are co-responsible for fast excitatory or inhibitory neurotransmission. Due to this crucial physiological role, dysfunction of pLGICs can lead to severe disorders, including epilepsy, myasthenia gravis and Alzheimer’s disease. For the development of new drugs to treat these and other diseases, a detailed insight in the structure of pLGICs is highly useful. However, it is a difficult task to determine the structure of ion channels. Nowadays, this is usually achieved through X-ray crystallography. In order to apply this technique, ion channels need to be extracted out of their lipid rich environment. This often results in proteins that are no longer biochemically stable and thus unsuitable for structural studies. During this PhD project we searched for pLGICs which behave biochemically favorable. We succeeded in identifying both a prokaryotic and a eukaryotic pLGIC that exhibit distinct biochemical stability.
Firstly, we identified CyLIC, a prokaryotic homologue derived from Cyanothece sp. PCC 7425. In contrast to ELIC and GLIC, prokaryotic, cation-selective homologues with known three-dimensional structure, CyLIC appeared to be anion-selective based upon sequence analysis. However, electrophysiological experiments did not reveal any activating ligands, excluding any further functional characterization. Large-scale purification of CyLIC, consisting of heterologous expression, membrane isolation, detergent solubilization, affinity chromatography and gelfiltration, resulted in pure and monodisperse, oligomeric CyLIC. Crystallization of this purified protein lead to a large amount of protein crystals in varying conditions. However, their diffraction quality was limited due to anisotropy. To overcome this problem, we have implemented the use of Fab fragments as crystallization chaperones. A screen was performed to identify Fab fragments that bind CyLIC with a high stoichiometry, maximally expanding the available surface area for the formation of crystal contacts. These identified Fab fragments were subsequently used for co-crystallization trials, but a decrease in overall resolution limit was observed and the anisotropic diffraction remained, though to a lesser extent. Therefore, we turned our attention towards another technique for structure determination, single particle cryo-EM. These experiments allowed us to reconstruct a first three-dimensional model of CyLIC. However, the resolution was limited to 12 Å due to the heterogeneous angular distribution.
Secondly, we performed a BLAST search in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid thriving in the vicinity of hydrothermal vents. It has been shown that proteins from extremophilic organisms display superior stability under laboratory conditions, making them ideal candidates for structural studies. We identified seven pLGIC homologues and named them Alpo1-7. In this PhD project, we mainly focused on Alpo1. Sequence analysis of Alpo1 revealed the presence of conserved residues and lead to first hypotheses concerning functionality. We have attempted to confirm these hypotheses by electrophysiological experiments but did not succeed in identifying activating ligands. However, we continued with the biochemical characterization of Alpo1. Alpo1 was covalently coupled to eGFP, Alpo1-eGFP, and heterologously expressed in Sf9 insect cells whereafter FSEC-experiments were conducted to identify suitable detergents for extraction of this membrane protein from the lipid bilayer. Strikingly, Alpo1-eGFP could be extracted in a monodisperse, oligomeric state by practically all detergents tested. Subsequent large-scale purification of Alpo1-eGFP and Alpo1-wt resulted in milligram amounts of pure and oligomeric protein. Additionally, we could demonstrate that Alpo1 indeed exhibited an exceptionally high melting temperature as was expected based upon its extremophilic origin. Notwithstanding this apparent stability, no protein crystals could be obtained. Therefore we decided to produce Nanobodies directed against Alpo1. One particular Nanobody, Nb9, exhibited the highest binding stoichiometry and increased the resistance of Alpo1 to heating. We have used this Nanobody for co-crystallization trials, but did not obtain any protein crystals. Furthermore, we have designed a truncated construct, lacking the intracellular M3-M4 loop and have performed both limited proteolysis and deglycosylation experiments. However, the results of these experiments are rather preliminary.
Overall, no high-resolution structural information was obtained despite the implementation of various strategies. However, we are convinced that the extensive biochemical characterization, described in this doctoral thesis, can result in future, successful structural studies of pLGICs.
ISBN: 978 94 6165 193 8
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Structural Neurobiology
Toxicology and Pharmacology

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