Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

Preparation, characterization and intramolecular electron-transfer studies of ruthenium-modified cytochromes c

Redox-active ruthenium complexes have been covalently attached to the surface of a series of natural, semisynthetic and recombinant cytochromes c. The protein derivatives were characterized by a variety of spectroscopic techniques. Distant Fe^(2+) - Ru^(3+) electronic couplings were extracted from intramolecular electron-transfer rates in Ru(bpy)_2(im)HisX (where X= 33, 39, 62, and 72) derivatives of cyt c. The couplings increase according to 62 (0.0060) < 72 (0.057) < 33 (0.097) < 39 (0.11 cm^(-1)); however, this order is incongruent with histidine to heme edge-edge distances [62 (14.8) > 39 (12.3) > 33 (11.1) > =72 (8.4 Å)]. These results suggest the chemical nature of the intervening medium needs to be considered for a more precise evaluation of couplings. The rates (and couplings) correlate with the lengths of a-tunneling pathways comprised of covalent bonds, hydrogen bonds and through-space jumps from the histidines to the heme group. Space jumps greatly decrease couplings: one from Pro71 to Met80 extends the σ-tunneling length of the His72 pathway by roughly 10 covalent bond units. Experimental couplings also correlate well with those calculated using extended Hiickel theory to evaluate the contribution of the intervening protein medium.

Two horse heart cyt c variants incorporating the unnatural amino acids (S)-2- amino-3-(2,2'-bipyrid-6-yl)-propanoic acid (6Bpa) and (S)-2-amino-3-(2,2'-bipyrid-4-yl)propanoic acid ( 4Bpa) at position 72 have been prepared using semisynthetic protocols. Negligible perturbation of the protein structure results from this introduction of unnatural amino acids. Redox-active Ru(2,2'-bipyridine)_2^(2+) binds to 4Bpa72 cyt c but not to the 6Bpa protein. Enhanced ET rates were observed in the Ru(bpy)_2^(2+)-modified 4Bpa72 cyt c relative to the analogous His72 derivative. The rapid (< 60 nanosecond) photogeneration of ferrous Ru-modified 4Bpa72 cyt c in the conformationally altered alkaline state demonstrates that laser-induced ET can be employed to study submicrosecond protein-folding events.

Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.

Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural α-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.

In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased β-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.

Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and α4β4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the α4β4 receptor.

Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the α7 nAChR. Additionally, binding interactions to the complementary face of the α7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.

Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.

Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.