Non-Canonical Amino Acids As Biochemical Probes of Ligand-Gated Ion Channel Structure and Function

This dissertation describes several different chemical-scale studies of proteins involved in cellular signaling. The primary focus of this work is on ligand-gated ion channels, an important family of membrane receptors. In each study, the incorporation of structurally diverse non-canonical amino acids have been used to attain a high level of precision in probing the mechanisms of molecular processes. The first chapter provides an introduction to the nonsense suppression methodology used to genetically encode these probes, and surveys the classes of proteins studied herein. The second and third chapters are concerned with the mechanism of activation of a prokaryotic receptor, Gloeobacter violaceus ligand-gated ion channel. In these experiments, novel histidine derivatives were designed, synthesized, and incorporated to test the functional importance of acid-base titration at several positions in the receptor. Then, a battery of proline analogs were used to identify necessary structural features of several critical proline residues, providing clues to conformational changes that occur during receptor activation. The fourth chapter discusses studies of a different class of receptors, the Acid-Sensing Ion Channels. Several fluorinated aspartic acid and glutamic acid derivatives were targeted to modulate the acidity of putative proton binding sites. Attempts at preparation of these compounds for incorporation into proteins are detailed. Additional sections describe, efforts to elucidate factors in the binding selectivity of the tarantula venom psalmotoxin and selectivity of cation permeability. In the final chapter, early efforts at developing crosslinking assays for protein-protein interactions in mammalian cells are outlined. These assays involve the introduction of orthogonal tRNA/synthetase pairs for genetically encoding photoreactive phenylalanine analogs. The most successful studies involve the ligand-dependent dimerization of the soluble nuclear receptor, estrogen receptor α. However, the ultimate goal of this work is to to probe protein-protein interactions among membrane receptors and other proteins. Progress toward extension of the photocrosslinking assay into membrane receptors is described.