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Functional Evaluation of Noncovalent Interactions in Neuroreceptors and Progress Toward the Expansion of Unnatural Amino Acid Methodology


Daeffler, Kristina Nicole-McCleary (2014) Functional Evaluation of Noncovalent Interactions in Neuroreceptors and Progress Toward the Expansion of Unnatural Amino Acid Methodology. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ST7S-DB65.


This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.

In chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-π interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-π interaction with dopamine.

Chapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.

Chapter 4 identifies a cation-π interaction between glutamate and a tyrosine residue on loop C in the GluClβ receptor. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.

Chapter 5 examines inherent properties of the GluClα receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Unnatural amino acid, GPCR, Cys-loop receptor, dopamine, glutamate
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dougherty, Dennis A.
Thesis Committee:
  • Barton, Jacqueline K. (chair)
  • Heath, James R.
  • Rees, Douglas C.
  • Dougherty, Dennis A.
Defense Date:2 May 2014
Funding AgencyGrant Number
National Institutes of HealthGM081662
National Institutes of HealthNS034407
Record Number:CaltechTHESIS:05042014-135648744
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 2
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8212
Deposited By: Kristina Daeffler
Deposited On:16 May 2014 22:40
Last Modified:04 Oct 2019 00:04

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