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

by Brandt, Gabriel Shaw

Abstract (Summary)
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.
Bibliographical Information:

Advisor:John E. Bercaw; Henry A. Lester; Peter B. Dervan; Dennis A. Dougherty; Douglas C. Rees

School:California Institute of Technology

School Location:USA - California

Source Type:Master's Thesis



Date of Publication:10/04/2002

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