Cuvette and SEC-SANS data of GLIC from ILL 8-03-959

Raw SANS data from GLIC collected during experiment ILL 8-03-959, with DOI: https://doi.ill.fr/10.5291/ILL-DATA.8-03-95

Combining low resolution, high resolution, functional, and simulation techniques : In the study of pentameric ligand-gated ion channels

Combining methods yielding different information is a powerful approach for understanding structural biology in general, and in particular systems such as pentameric ligand-gated ion channels (pLGICs). Pentameric ligand-gated ion channels are membrane proteins that sense chemical signals, which they convert to changes in membrane potential.  pLGICs constitute important drug targets in humans, for example as sites of action for general anaesthetics. There are bacterial homologs, of which some - like the Gloeobacter violaceus ligand-gated channel GLIC - are suitable model systems, while others - like DeCLIC from a Desulfofustis deltaproteobacterium - demonstrate the structural range of this protein family. This thesis presents how low resolution, high resolution, functional, and simulation techniques have been combined in the study of GLIC and DeCLIC. The methods covered are small-angle neutron scattering (low resolution structural method), cryogenic electron microscopy (high resolution structural method), electrophysiology (functional method), and molecular dynamics simulations (simulation method), with a particular focus on the scattering and simulation experiments.  In the presented work, simulations and functional experiments are combined to elucidate modulation of GLIC by general anaesthetics, the average solution structure of both GLIC and DeCLIC is described from small-angle scattering utilizing conformational sampling from simulations, and new conformations of DeCLIC are found through cryogenic electron microscopy - including an open conformation consistent with scattering under corresponding solution conditions. This work has contributed to both the understanding of complex allosteric modulation, and of the conformational range available to pentameric ligand-gated ion channels. On the methodological side, I have furthered approaches for integrating small-angle neutron scattering and molecular dynamics simulations to describe membrane protein structure in solution - which stands to increase the information gained from scattering experiments, and to promote the use of scattering as a complementary technique in structural studies

Combining low resolution, high resolution, functional, and simulation techniques : In the study of pentameric ligand-gated ion channels

Combining methods yielding different information is a powerful approach for understanding structural biology in general, and in particular systems such as pentameric ligand-gated ion channels (pLGICs). Pentameric ligand-gated ion channels are membrane proteins that sense chemical signals, which they convert to changes in membrane potential.  pLGICs constitute important drug targets in humans, for example as sites of action for general anaesthetics. There are bacterial homologs, of which some - like the Gloeobacter violaceus ligand-gated channel GLIC - are suitable model systems, while others - like DeCLIC from a Desulfofustis deltaproteobacterium - demonstrate the structural range of this protein family. This thesis presents how low resolution, high resolution, functional, and simulation techniques have been combined in the study of GLIC and DeCLIC. The methods covered are small-angle neutron scattering (low resolution structural method), cryogenic electron microscopy (high resolution structural method), electrophysiology (functional method), and molecular dynamics simulations (simulation method), with a particular focus on the scattering and simulation experiments.  In the presented work, simulations and functional experiments are combined to elucidate modulation of GLIC by general anaesthetics, the average solution structure of both GLIC and DeCLIC is described from small-angle scattering utilizing conformational sampling from simulations, and new conformations of DeCLIC are found through cryogenic electron microscopy - including an open conformation consistent with scattering under corresponding solution conditions. This work has contributed to both the understanding of complex allosteric modulation, and of the conformational range available to pentameric ligand-gated ion channels. On the methodological side, I have furthered approaches for integrating small-angle neutron scattering and molecular dynamics simulations to describe membrane protein structure in solution - which stands to increase the information gained from scattering experiments, and to promote the use of scattering as a complementary technique in structural studies

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources