Defense Date

3-25-2016

Graduation Date

Spring 1-1-2016

Availability

Worldwide Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Michael Cascio

Committee Member

Jeffry Madura

Committee Member

Stephanie Wetzel

Committee Member

Jana Patton-Vogt

Keywords

Crosslinking, GlyR, Mass Spectrometry, Membrane Proteins

Abstract

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in the presence of selected ligands: resting (no ligand), desensitized (saturating glycine) and open state (non-desensitizing ivermectin (IVM)-gated F207A/A288G GlyR). Photo-crosslinking methodology linked with mass spectrometric analysis was developed on systematically generated single Cys mutations in GlyR with both Cys null and IVM sensitive backgrounds to enable the study of state-dependent structures of GlyR in comparative crosslinking studies. Studies were conducted on A41C and H419C mutants. A41 is shown to be in proximity to the pre-M1 and the M2-M3 loop region crucial for gating. Prior to these studies, very little information on H419 was available as it is located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other related pentameric ligand-gated ion channels. These studies identified specific GlyR crosslinks unique to each conformational state and identified potential motions in the receptor upon gating and desensitization. The defined distance constraints will be used to update our model of human α1-GlyR and provide insight into channel function. Significantly, this methodological approach is amenable to study any allosteric protein and complement other high resolution structural studies in identifying protein dynamics.

Format

PDF

Language

English

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