Using Crosslinking And Mass Spectrometry To Study Glycine Receptor Allostery

A wealth of high-resolution structural information regarding pentameric ligand-gated ion channels is now available, but less is known of the molecular details underlying complex allosteric mechanisms involved in channel gating and desensitization. Receptor allostery can be studied by identifying state-dependent distance constraints that may be used in molecular modeling of these receptors. Systematically generated single Cys mutations of the human α1 glycine receptor (GlyR) expressed in insect cells were labeled with a clickable methanethiosulfonate-benzophenone crosslinker. After covalent ligation to Cys, crosslinks may then be introduced in the resting, open, or desensitized states by photoactivation. Including an alkyne tag on the crosslinker permits click chemistry addition of biotin, which allows for enrichment by avidin chromatography. Mass spectrometry (MS) fingerprinting of monomeric and higher-order GlyR bands on SDS-PAGE using ESI-QTOF MS/MS then allows for determination of the site of crosslinking. Our initial proof-of-principle studies conducted on purified GlyR have provided state-dependent information on this receptor. This approach may be broadly applicable to studies of any allosteric complex.