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The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are required. This dissertation demonstrates a novel complex utilizing earth-abundant Ni in combination with glycine as an efficient catalyst with a modest overpotential of 0.475 ± 0.005 V for a current density of 1 mA/cm2 at pH 11. The production of molecular oxygen at a high potential was verified by measurement of the change in oxygen concentration, yielding a Faradaic efficiency of 60 ± 5%. This Ni species can achieve a current density of 4 mA/cm2 that persists for at least 10 hours. Based upon the observed pH dependence of the current amplitude and oxidation/reduction peaks, the catalysis is an electron-proton coupled process. In addition, to investigate the binding of divalent metals to proteins, four peptides were designed and synthesized with carboxylate and histidine ligands. The binding of the metals was characterized by monitoring the metal-induced changes in circular dichroism spectra. Cyclic voltammetry demonstrated that bound copper underwent a Cu(I)/Cu(II) oxidation/reduction change at a potential of approximately 0.32 V in a quasi-reversible process. The relative binding affinity of Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) to the peptides is correlated with the stability constants of the Irving-Williams series for divalent metal ions. A potential application of these complexes of transition metals with amino acids or peptides is in the development of artificial photosynthetic cells.
ContributorsWang, Dong (Author) / Allen, James P. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
Description
Carbonaceous chondrites (CCs) present a unique opportunity for learning about the earliest organic chemistry that took place in our Solar System. The complex and diverse suite of meteoritic organic material is the result of multiple settings and physicochemical processes, including aqueous and thermal alteration. Though meteorites often inform origin-of-life discussions because they could have seeded early Earth with significant amounts of water and pre-biotic, organic material, their record of abiotic, aqueous, and organic geochemistry is of interest as well.
CC materials previously resided on asteroidal parent bodies, relic planetesimals of Solar System formation which never accreted enough material to develop long-lived, large-scale geological processes. These bodies were large enough, however, to experience some degree of heating due to the decay of radiogenic isotopes, and the meteorite record suggests the existence of 100-150 parent bodies which experienced varying degrees of thermal and aqueous alteration for the first several 10 Myr of Solar System history.
The first chapter of this dissertation reviews literature addressing aqueous alteration as an essential participant in parent body geochemistry, organic synthesis, or both (though papers which address both are rare). The second chapter is a published organic analysis of the soluble organic material of Bells, an unclassified type 2 chondrite. Analytical approaches to assess terrestrial contamination of meteorite samples are also reviewed in the first chapter to allow introduction in chapter 3 of kinetic modeling which rules out certain cases of contamination and constrains the timing of thermal and aqueous alteration. This is the first known application of isoleucine epimerization for either of these purposes. Chapter 4 is a kinetic study of D-allo-isoleucine epimerization to establish its behavior in systems with large, relative abundances of alloisoleucine to isoleucine. Previous epimerization studies for paleontological or geological purposes began with L-isoleucine, the only protein amino acid of the four isoleucine stereoisomers.
Kinetic model calculations using isoleucine stereoisomer abundances from 7 CR chondrites constrain the total duration of the amino acids' residence in the aqueous phase. The comparatively short timescales produced by the presented modeling elicit hypotheses for protection or transport of the amino acids within the CR parent body.
CC materials previously resided on asteroidal parent bodies, relic planetesimals of Solar System formation which never accreted enough material to develop long-lived, large-scale geological processes. These bodies were large enough, however, to experience some degree of heating due to the decay of radiogenic isotopes, and the meteorite record suggests the existence of 100-150 parent bodies which experienced varying degrees of thermal and aqueous alteration for the first several 10 Myr of Solar System history.
The first chapter of this dissertation reviews literature addressing aqueous alteration as an essential participant in parent body geochemistry, organic synthesis, or both (though papers which address both are rare). The second chapter is a published organic analysis of the soluble organic material of Bells, an unclassified type 2 chondrite. Analytical approaches to assess terrestrial contamination of meteorite samples are also reviewed in the first chapter to allow introduction in chapter 3 of kinetic modeling which rules out certain cases of contamination and constrains the timing of thermal and aqueous alteration. This is the first known application of isoleucine epimerization for either of these purposes. Chapter 4 is a kinetic study of D-allo-isoleucine epimerization to establish its behavior in systems with large, relative abundances of alloisoleucine to isoleucine. Previous epimerization studies for paleontological or geological purposes began with L-isoleucine, the only protein amino acid of the four isoleucine stereoisomers.
Kinetic model calculations using isoleucine stereoisomer abundances from 7 CR chondrites constrain the total duration of the amino acids' residence in the aqueous phase. The comparatively short timescales produced by the presented modeling elicit hypotheses for protection or transport of the amino acids within the CR parent body.
ContributorsMonroe, Adam Alexander (Author) / Pizzarello, Sandra (Thesis advisor) / Williams, Peter (Thesis advisor) / Anbar, Ariel D (Committee member) / Shock, Everett L (Committee member) / Arizona State University (Publisher)
Created2014
Description
The synthesis of the bis(2-diphenylphosphinoethyl)amine chelating ligand (1) was a crucial component in the preparation of non-canonical amino acids (NCAAs) throughout the project. Studies in this project indicated the need to isolate the ligand from its hydrochloride salt form seen in (1) which led to the synthesis of the brown oil, (Ph2PCH2CH2)2NH, (2). The ligand features a phosphine-nitrogen-phosphine group that is not observed in existing NCAAs. Phosphine groups are rarely seen in existing NCAAs and avoided by biochemists because they tend to oxidize before metal addition. In this project, (1) was used in a 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) mediated method and palladium-catalyzed method to tether an amino acid to the nitrogen atom of the ligand framework. Both methods were monitored through the use of Nuclear Magnetic Resonance (NMR) spectroscopy. While the palladium catalyzed method exhibited little to no coupling, the 31P NMR spectrum obtained for the HATU mediated method did reveal that some coupling had occurred. The unsuccessful attempts to tether an amino acid to (1) led to the hypothesis that the phosphine groups were interfering with the palladium catalyst during the cross-coupling reaction. In an effort to test this hypothesis, (2) was reacted with the dimer, [Rh(nbd)Cl]2, to coordinate the rhodium metal to the free phosphorous arms and the nitrogen atom of the isolated PNP ligand. The PNP-based metal complex was used in the palladium catalyzed method, but cross-coupling was not observed. The new PNP-based metal complex was investigated to demonstrate that it exhibits moisture and air stability.
ContributorsManjarrez, Yvonne (Author) / Trovitch, Ryan (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Herckes, Pierre (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Continuing and increasing reliance on fossil fuels to satisfy our population’s energy demands has encouraged the search for renewable carbon-free and carbon-neutral sources, such as hydrogen gas or CO2 reduction products. Inspired by nature, one of the objectives of this dissertation was to develop protein-based strategies that can be applied in the production of green fuels. The first project of this dissertation aimed at developing a controllable strategy to incorporate domains with different functions (e. g. catalytic sites, electron transfer modules, light absorbing subunits) into a single multicomponent system. This was accomplished through the rational design of 2,2’-bipyridine modified dimeric peptides that allowed their metal-directed oligomerization by forming tris(bipyridine) complexes, thus resulting in the formation of a hexameric assembly.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.
ContributorsAlcala-Torano, Rafael de Jesus (Author) / Ghirlanda, Giovanna (Thesis advisor) / Moore, Ana L (Committee member) / Mills, Jeremy H (Committee member) / Arizona State University (Publisher)
Created2019
Description
Non-canonical amino acids (NCAAs) can be used in protein chemistry to determine their structures. A common method for imaging proteins is cryo-electron microscopy (cryo-EM) which is ideal for imaging proteins that cannot be obtained in large quantities. Proteins with indistinguishable features are difficult to image using this method due to the large size requirements, therefore antibodies designed specifically for binding these proteins have been utilized to better identify the proteins. By using an existing antibody that binds to stilbene, NCAAs containing this molecule can be used as a linker between proteins and an antibody. Stilbene containing amino acids can be integrated into proteins to make this process more access able. In this paper, synthesis methods for various NCAAs containing stilbene were proposed. The resulting successfully synthesized NCAAs were E)-N6-(5-oxo-5-((4-styrylphenyl) amino) pentanoyl) lysine, (R,E)-2-amino-3-(5-oxo-5-((4-styrylphenyl)amino)pentanamido)propanoic acid, (E)-2-amino-5-(5-oxo-5-((4-styrylphenyl) amino) pentanamido) pentanoic acid. A synthesis for three more shorter amino acids, (R,E)-2-amino-3-(3-oxo-3-((4-styrylphenyl) amino) propanamido) propanoic acid, (E)-2-amino-5-(3-oxo-3-((4-styrylphenyl) amino) propanamido) pentanoic acid, and (E)-N6-(3-oxo-3-((4-styrylphenyl) amino) propanoyl) lysine, is also proposed.
ContributorsJenkins, Bryll (Author) / Mills, Jeremy (Thesis director) / Ghirlanda, Giovanna (Committee member) / Nannenga, Brent (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05
Description
Glioblastoma brain tumors are among the most lethal human cancers. Treatment efforts typically involve both surgical tumor removal, as well as ongoing therapy. In this work, we propose the use of deuterium magnetic resonance imaging (MRI) to delineate tumor boundaries based on spatial distributions of deuterated leucine, as well as resolve the metabolism of leucine within the tumor. Accurate boundary identification contributes to effectiveness of tumor removal efforts, while amino acid metabolism information may help characterize tumor malignancy and guide ongoing treatment. So, we first examine the fundamental mechanisms of deuterium MRI. We then discuss the use of spin-echo and gradient recall echo sequences for mapping spatial distributions of deuterated leucine, and the use of single-voxel spectroscopy for imaging metabolites within a tumor.
ContributorsCostelle, Anna (Author) / Beeman, Scott (Thesis director) / Kodibagkar, Vikram (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2022-05
Description
Different populations of evolved E.coli and their ancestors were grown in a variety of single amino acid environments to determine their ability to use that amino acid as a carbon source. Some evolved lines were able to grow in amino acids that their ancestors weren't able to. The source of this change in amino acid growth was investigated by testing uptake, searching for candidate mutations, and comparing growth rates of populations with and without certain mutations.
ContributorsKing, Lily (Author) / Ho, Wei-Chin (Thesis director) / Lynch, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05