1-Oxo-5-hydroxytryptamine: A Surprisingly Potent Agonist of the 5-HT_3(Serotonin) Receptor

A novel synthetic route to 1-oxo-5-hydroxytryptamine, the benzofuran analogue of serotonin, has been developed. The new synthesis proceeds via the [3+2] cycloaddition of p-benzoquinone and 2,3-dihydrofuran, followed by a Lewis acid-catalyzed isomerization. This molecule proves to be a competent agonist (equipotent to serotonin) of the 5-HT_3 receptor, demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor

A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT3 Receptor†

The binding sites of Cys-loop receptors are formed from at least six loops (A−F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R_(max)) 2−19-fold, and decreased n_H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [^3H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT_3 Receptor

The binding sites of Cys-loop receptors are formed from at least six loops (A−F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R_(max)) 2−19-fold, and decreased n_H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [^3H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available

Chemical-Scale Studies of the 5-HT₃ and D2 Dopamine Receptors

During synaptic transmission in the central nervous system, neuroreceptors transduce a chemical signal into an electrical signal, a process that is mediated by both ligand-gated ion channels (LGICs) and G-protein coupled receptors (GPCRs). The work in this thesis examines structure-function relationships within these receptors, with a focus on elucidating the mechanism of molecular recognition during ligand binding. We utilize conventional and unnatural amino acid mutagenesis, structural derivatives of agonists, and homology models to identify specific interactions and the role of binding site residues in ligand binding and receptor activation. The technique of unnatural amino acid mutagenesis allows us to study these processes in greater detail than would otherwise be possible, even at the scale of a chemical bond. Chapter 2 covers structure-function investigations of a ligand-gated ion channel, the 5-HT₃ receptor, with a goal of understanding agonist binding and receptor activation. The project examines residues in close proximity to the ligand-binding site and focuses on polar interactions with hydrophilic residues. We identify 5-fluorotryptamine (5-FT) as a partial agonist of the 5-HT₃ receptors and show that size and electronegativity are important at the 5’ position for efficient channel opening. Our investigation of the compound 1-OT revealed it to be an agonist of equal potency to the native agonist (5-HT), demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor. A study focusing on loop A residues led us to refine our homology model and propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Further studies of binding site residues identified an ionic interaction that likely participates in the conformational changes associated with receptor gating and characterized several other residues that play critical roles in receptor activation. Finally, we compare and contrast the behaviors of two structurally distinct agonist classes, 5-HT and its related structures, and m-chlorophenylbiguanide (mCPBG) and identify several residues that play critical roles in modulating agonist binding and gating in response to these agonists. Chapter 3 describes a study examining the binding site and the mechanism of agonist activation of a GPCR, the D2 dopamine receptor. A number of aromatic amino acids thought to be near the agonist binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at a conserved tryptophan of the D2 receptor establishes a cation-π interaction between the agonist dopamine and this residue (W6.48), suggesting a reorientation of W6.48 on agonist binding, consistent with proposed "rotamer switch" models. Finally, chapter 4 describes a project that seeks to extend the nonsense suppression methodology to include mammalian expression systems. Progress is made developing techniques for efficient transfection of cells in culture.</p

Synergistic Effects of Pesticides and Metals on The Fibrillation of α-synuclein: Implications for Parkinson’s Diseasev

Aggregation of α-synuclein has been implicated in the formation of proteinaceous inclusions in the brain (Lewy bodies, Lewy neurites) that are characteristic of neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs). The etiology of PD is unknown, but recent work has shown that except in rare cases, there appears to be no direct genetic basis. However, several studies have implicated environmental factors, especially pesticides and metals. Here we show that certain pesticides and metals induce a conformational change in α-synuclein and directly accelerate the rate of formation of α-synuclein fibrils in vitro. In addition, the simultaneous presence of metal and pesticide led to synergistic effects on the rate of fibrillation. We propose a model in which environmental factors in conjunction with genetic susceptibility may form the underlying molecular basis for idiopathic PD

A Coupled Array of Noncovalent Interactions Impacts the Function of the 5‑HT_3A Serotonin Receptor in an Agonist-Specific Way

The serotonin type 3A (5-HT_3A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation−π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation−π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation−π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating

Accelerated α-synuclein Fibrillation in Crowded Milieu

Parkinson\u27s disease is the second most common age-related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α-synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/l. To address this discrepancy, the effect of molecular crowding on α-synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethylene glycols) dramatically accelerated α-synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume