Defense Date

12-10-2020

Graduation Date

Spring 5-2021

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Michael Cascio

Committee Member

Mihaela-Rita Mihailescu

Committee Member

Michael J. Van Stipdonk

Committee Member

Benedict J. Kolber

Keywords

pLGICs, glycine receptors, crosslinking mass spectrometry

Abstract

A network of site-specific single Cys-mutations coupled with CX-MS can be used to elucidate a more refined structure of GlyR and obtain a more definitive understanding of pentameric ligand-gated ion channel (pLGIC) allostery. Each Cys-mutant is introduced into an a1 homomeric Cys null background (C41S/C290A/C345S), or in the same background with F207G/A288G mutation that allows non-desensitizing GlyR activation by ivermectin (IVM). State-dependent crosslinking with methanethiosulfonate benzophenone (MTS-bzp) to a single thiol of purified, vesicle reconstituted GlyR are conducted after enriching the receptor in different allosteric states: resting (no ligand), open (F207G/A288G + IVM), or desensitized (excess glycine). Digested peptides are analyzed via liquid chromatography mass spectrometry to identify sites of intra- and intermolecular crosslinking. Tandem MS of mass-shifted precursor ions further refine these distance constraints. Independent comparative studies targeting different single Cys GlyR (M287C, K116C, K206C) provides evidence of allosteric changes between the three states, as well as direct topological information of regions that are unresolved in other high-resolution structures of pentameric ligand gated ion channels (pLGICs), most notably the M3-M4 loop. The unresolved loop is shown to interact with the rest of the protein primarily in non-conducting states, possibly highlighting its role in structure in those states. Regions of receptor mobility have also been identified and are both novel and congruent with what is reported in literature. Reported mobile regions include the M2-M3 loop, the C-loop, M3-M4 loop and very near the N-terminus. These findings can be applied to further refinement of GlyR structure, and provide information on GlyR molecular mechanism. Additionally, methods reported in this work can also be applied to the understanding of other members of the pLGIC family, and other transmembrane proteins that are waiting further structural information.

Language

English

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