Author ORCID Identifier

0000-0002-2690-7140

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Vasanthi Jayaraman, Ph.D.

Committee Member

Jaroslaw Aronowski, M.D, Ph.D.

Committee Member

Alemayehu Gorfe, Ph.D.

Committee Member

Ruth Heidelberger, M.D., Ph.D.

Committee Member

John Spudich, Ph.D.

Abstract

The N-methyl-D-aspartate (NMDA) receptor is one member of a class of proteins known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, with the NMDA receptor standing out among these receptors for its requirement of a co-agonist, its magnesium-block-based coincidence detection, its slow kinetics, its calcium permeability, its allosteric modulation, and its especially important functional roles in synaptic plasticity, excitotoxicity, and more. In recent years, a wealth of structural information has come about describing endpoint structures to high resolution, but such structures are unable to fully resolve the movements and dynamics necessary for appropriate function. The work in this dissertation uses single molecule Förster Resonance Energy Transfer (smFRET) as a means to address that gap. We have examined the question of partial agonism of the NMDA receptor, noting a mechanism of a dynamically graded cleft closure. We have pushed the bounds of the temporal resolution of such methods and been able to resolve fast dynamics of the ligand-binding domain, noting the adherence of the domain to the conformational selection model, and the revelation of a novel conformation leading to activation hitherto unknown. Finally, we have also directly examined the conformational dynamics of the transmembrane domain of the NMDA receptor with regards to its gating motions, granting unprecedented insight into the movements of the ion channel domain and elucidating a novel mechanism of allosteric inhibition. Such biophysical characterization of the NMDA receptor is essential, not only simply to know how the receptor works, but also to develop effective therapeutics that do not impair the receptor’s important physiological roles.

Keywords

FRET, Glutamate Receptors, Biophysics, Conformational Selection, Protein Dynamics, Ligand-Gated Ion Channels, Membrane Proteins, Fluorescence, Spectrsocopy, Single Molecule

Available for download on Thursday, October 26, 2017

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