Functional evaluation of noncovalent interactions in neuroreceptors and progress toward the expansion of unnatural amino acid methodology

This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.

In chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-π interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-π interaction with dopamine.

Chapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.

Chapter 4 identifies a cation-π interaction between glutamate and a tyrosine residue on loop C in the GluClβ receptor. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.

Chapter 5 examines inherent properties of the GluClα receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Part I. Synthesis of L-amino acid oxidase by a serine- or glycine-requiring strain of neurospora. Part II. Studies concerning multiple electrophoretic forms of tyrosinase in neurospora

Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora

Wild-type cultures of Neurospora crassa growing on minimal medium contain low levels of L-amino acid oxidase, tyrosinase, and nicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes are derepressed by starvation and by a number of other conditions which are inhibitory to growth. L-amino acid oxidase is, in addition, induced by growth on amino acids. A mutant which produces large quantities of both L-amino acid oxidase and NADase when growing on minimal medium was investigated. Constitutive synthesis of L-amino acid oxidase was shown to be inherited as a single gene, called P110, which is separable from constitutive synthesis of NADase. P110 maps near the centromere on linkage group IV.

L-amino acid oxidase produced constitutively by P110 was partially purified and compared to partially purified L-amino acid oxidase produced by derepressed wild-type cultures. The enzymes are identical with respect to thermostability and molecular weight as judged by gel filtration.

The mutant P110 was shown to be an incompletely blocked auxotroph which requires serine or glycine. None of the enzymes involved in the synthesis of serine from 3-phosphoglyceric acid or glyceric acid was found to be deficient in the mutant, however. An investigation of the free intracellular amino acid pools of P110 indicated that the mutant is deficient in serine, glycine, and alanine, and accumulates threonine and homoserine.

The relationship between the amino acid requirement of P110 and its synthesis of L-amino acid oxidase is discussed.

Part II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora

Supernumerary bands shown by some crude tyrosinase preparations in paper electrophoresis were investigated. Genetic analysis indicated that the location of the extra bands is determined by the particular T allele present. The presence of supernumerary bands varies with the method used to derepress tyrosinase production, and with the duration of derepression. The extra bands are unstable and may convert to the major electrophoretic band, suggesting that they result from modification of a single protein. Attempts to isolate the supernumerary bands by continuous flow paper electrophoresis or density gradient zonal electrophoresis were unsuccessful.

The protein, peptide and free amino-acid contents of some species of Gracilaria from south-east coast of India

Four species of Gracilaria are investigated for their free amino-acid contents, as well as amino-acid constituents in the proteins and the peptides, using quantitative paper chromatographic technique. Amino-acid constituents of different species of Gracilaria differ only in amount, while free amino-acids and the amino-acids in the peptides vary both in quality and quantity. A number of amino-acids recorded as protein constituents have even escaped detection in the peptides, while in the free state they occur either in all the species or in some only except homocystine. Moreover, some amino-acids occur exclusively in the free state.

The free and combined amino acid contents in species of Caulerpa from southeast coast of India

Caulerpa racemosa var. macrophysa, C. racemosa var cornyphora and C. scalpelliformis are analyzed by quantitative paper chromatographic technique for their amino acid contents in proteins, peptides and free state. It is found that no appreciable variation occurs in the quality of amino acid make up in these algae; but quantitative differences are apparent in them. Moreover, both qualitative and quantitative variations occur in the amino acid make up of the peptides, and in the free state. The results are compared with those of the other investigations.

Effect of copper, cadmium and Cu.Cd mixture on amino acid content in the poslarvae of penaeid shrimp, Penaeus monodon and P. penicillatus

This study document effects of short-term (96h) sublethal levels of copper, cadmium and their mixture on the amino acid composition of postlarvae of the penaeid shrimp, P.monodon and P.penicillatus . All experimental conditions were kept constant, temperature between 25-27•C and salinity 21-22 ppt. The estimated LD50 for Cu was 200 ug/L, for Cd 177.5 ug/L and for Cu.Cd mixture 250ug/L. In P. penicillatus at the same concentration of each metal, there was significant reduction in amino acid content, which was 8.01% higher than the control. Almost similar reduction in some amino acids was observed in P.monodon. At the maximum concentration of 400 ug/L, cadmium caused higher reduction in amino acid composition than did copper. Thus, amino acid composition may be regarded as a sensitive biochemical indicator of Cu and Cd toxicity because of the effect of these metals on protein synthesis, a signal of physiological stress in marine organisms subjected to heavy metal pollution.

Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity

A novel L-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. Fifty percentage cytotoxic concentrations (CC50) of TSV-LAO on C8166 cells were 24 and 390 nM in the absence or presence of catalase (400nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs. (C) 2003 Elsevier Inc. All rights reserved.

Molecular characterization of L-amino acid oxidase from king cobra venom

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet a

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had n

Purification and characterization of a new L-amino acid oxidase from Daboia russellii siamensis venom

A new L-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size

Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom

An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogenicity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resourse Q ion-exchange chromatography. TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD. The 24 N-terminal ammo acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further studies found that TM-LAO inhibited the growth of E. colt, S. aurues and B. dysenteriae. TM-LAO also showed cytotoxicity and platelet aggregation activity. All the biological activities were eliminated by catalase, a H2O2 scavenger. It shows that these biological effects are possibly due to the formation of H2O2 produced by TM-LAO.

The free alpha amino acid nitrogen contents of some common food fishes of Kakinada region, Andhra Pradesh

The moisture and free alpha amino nitrogen contents of some important food fishes and shell fishes of Kakinada region have been studied. Crustaceans and molluscs contain free alpha amino acids in quantities several times higher than all other aquatic animals examined in this study. Their probable role in the physiological activities of these animals has been discussed.

A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti