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Site-specific incorporation of synthetic amino acids into functioning ion channels

Citation

Brandt, Gabriel Shaw (2003) Site-specific incorporation of synthetic amino acids into functioning ion channels. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-01202003-221429

Abstract

Synthetic amino acids may be introduced into functioning proteins by means of nonsense suppression, using tRNA aminoacylated with unnatural amino acids. This technique can be extended to living cells through micro-injection of mRNA and tRNA into Xenopus laevis oocytes. Introduction of synthetic amino acids into proteins has been used, broadly, for three purposes. First, sensitive probes have been incorporated into proteins, using side chain chemistry unavailable to naturally encoded amino acids. Second, reactive side chains have been developed which can drive conformational rearrangements of the protein. Third, natural post-translational modifications of protein side chains have been mimicked. The work presented here applies all of these approaches to the study of ion channels. A series of fluorinated Trp residues was incorporated into the nicotinic acetylcholine receptor (nAChR) to probe electrostatic effects on cation-pi mediated binding of nicotine and other agonists. Site-specific protein backbone cleavage of the nicotinic acetylcholine and purinergic P2X2 receptors was undertaken, along with intersubunit photo-crosslinking in the nAChR. Caged tyrosine was employed to study tyrosine phosphorylation of an important modulatory site in the potassium channel Kir2.1. Finally, caged phosphoamino acid analogs were synthesized for further characterization of the effects of phosphorylation on ion channels.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:acetylcholine; caged compound; cation-pi; fluorine; molecular neurobiology; nicotine; nicotinic; nonsense suppression; phosphorylation; photochemistry; unnatural amino acid
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:
  • Rees, Douglas C. (chair)
  • Dougherty, Dennis A.
  • Lester, Henry A.
  • Bercaw, John E.
  • Dervan, Peter B.
Defense Date:4 October 2002
Author Email:gabrielb (AT) caltech.edu
Record Number:CaltechETD:etd-01202003-221429
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-01202003-221429
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:245
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:21 Jan 2003
Last Modified:26 Dec 2012 02:28

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