Analysis of Single-Site Cysteine Mutation, I412C, in Human A Glycine Receptor States to Further Refine Structure and Allostery
Date of this Version
Engquist, L. 2021. Analysis of Single-Site Cysteine Mutation, I412C, in Human A Glycine Receptor States to Further Refine Structure and Allostery. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
The glycine receptor (GlyR) is the major inhibitory receptor in the brain and spinal cord. A member of the pentameric ligand gated ion channel superfamily, crystal structures are available but there are still unresolved areas, specifically the C-terminal tail and TM3-TM4 intracellular loop. Further refinement can provide deeper understanding of the molecular mechanism and allow the creation of novel therapeutics to modulate its function. We propose to insert a single cysteine mutation, I412C, into a Cys null background (C41S/C290A/C345S) to study non- conducting states (resting, desensitized) or with F207G/A288G mutations to study the open state. Purified, reconstituted GlyR is crosslinked with a Cys-specific photoactivatable heterobifunctional crosslinker, methanethiosulfonate benzophenone, to produce state dependent crosslinks. After digestion, mass shifted peptides are analyzed via mass spectrometry to identify inter- or intracellular crosslinks. Initial results show a different crosslinking pattern between the three states, mapping dynamic changes in receptor structure upon channel gating and desensitization. Future work involves more trials refining these crosslinked peptides to single residue locations.
Copyright Leah Engquist 2021