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

Abstract

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.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs

“…Investigating lipid exchange enables the determination of thermodynamic quantities and provides a key comparative measure for different membrane protein carrier systems in terms of colloidal and structural stability as well as a direct insight to the dynamics of the lipids inside. , Here, we probed the lipid exchange by TR-SANS, , which monitors the decay of the scattered intensity as deuterated and hydrogenous lipids are exchanged between nanodiscs over time. More specifically, the initial signal is the sum of the scattering intensity from h-DMPC- and d-DMPC-loaded nanodiscs that decays to a background level over time after mixing the samples as lipids are randomly distributing between the nanodiscs.…”

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

“…SEC-SANS experiments [30] were performed at the D22 beamline of Institute Laue–Langevin, using a paused flow approach [6]. In short, the protein sample was loaded on a Superdex 200 Increase 10/300 gel filtration column in-line with the SANS measurement[31, 32], exchanging the sample to the D 2 O buffer environment and match-out deuterated DDM (d-DDM) [33] prior to the protein reaching the SANS measuring cell.…”