Examining Glycine Receptor's Intracellular Domain by Crosslinking-Mass Spectrometry

Project Summary. While high-resolution structural data are available for members of the pentameric ligand-gated ion channel (pLGIC) superfamily in membrane-mimetic environments, the structure of the intracellular domain (ICD) of these receptors in lipid bilayers remains unresolved. Our aims are to conduct crosslinking mass spectrometric studies (CXMS) to complement current methods and characterize the structure of full-length a1 glycine receptors (GlyRs) in reconstituted bilayers different allosteric states, with a particular focus on the ICD. Specific Aim 1 will use CXMS to interrogate the protein structure of the ICD around single sites of attachment of a variable-length photoactivatable protein-protein crosslinkers distributed over the full length of the ICD as a function of GlyR allostery. Specific Aim 2 will use CXMS to examine lipid-protein interactions as a function of allostery using lipid crosslinkers with their respective photoactivatable moieties at different depths of the bilayer, with a particular focus on the close associations of the ICD with the lipid bilayer. Together, the CXMS approaches used in each Aim will provide insight into the structure of this previously unresolved domain and complement high resolution studies conducted in membrane-mimetic environments.