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Amino acid transport in equine erythrocytes.January 1985 (has links)
by Daron Adam Fincham. / Bibliography: leaves [183]-[210] / Thesis (Ph.D.)--Chinese University of Hong Kong, 1985
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Biomimetic Aminoacylation: Optimization of Reaction ConditionsBunn, Shannon Elizabeth 05 January 2010 (has links)
Synthesizing proteins containing unnatural amino acids inserted at specific positions within the protein sequence has been a longstanding goal of biological chemists. This poses unique challenges, as aminoacyl tRNA synthetases, the enzymes responsible for protein synthesis, are highly specific. To overcome this, a lanthanum-catalyzed, biomimetic tRNA aminoacylation method has been developed(1). However, due to unproductive lanthanum coordination of ethyl phosphate, a reaction byproduct, a full equivalent of lanthanum must be added to each reaction. This may threaten the integrity of tRNA, as lanthanides are known to catalyze the hydrolysis of RNA (2, 3). Using uridine as a simplified tRNA mimic, magnesium, which is known to coordinate strongly with phosphate ions, has been utilized to optimize this reaction and increase the selectivity of lanthanum towards esterification. In the presence of magnesium, ester yield is substantially increased. In addition to this, optimal pH and buffer reaction conditions were determined.
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Biomimetic Aminoacylation: Investigating Detection of Acylation and the Effect of α-Amino ProtectionAndrusiak, Tara 15 December 2009 (has links)
Direct synthesis of aminoacyl-tRNA occurs using α-N-tBoc-protected aminoacyl phosphates and lanthanum salts. Deprotection of aminoacyl tRNA is essential prior to translation; however, the conditions of tBoc deprotection causes tRNA degradation. It was found that α-N-pentenoyl-protected aminoacyl phosphates, deprotected under mild conditions, are effectively used in lanthanum-mediated acylation of tRNA analogs. This provides an alternative route for aminoacyl-tRNA synthesis that maintains tRNA structure. Also, it was determined that α-N-deprotection of aminoacyl phosphates prior to aminoacylation still produces aminoacylated tRNA analogs. This establishes that acyl phosphates activate amino acids without inducing self-condensation, presumably due to electrostatic repulsion. Direct quantification of lanthanum-mediated tRNA aminoacylation was additionally undertaken utilizing a radiolabelled tRNA assay. From this, it was shown that lanthanum-mediated acylation does not promote deacylation and degradation of tRNA. These results have provided insight into lanthanum-mediated acylation of tRNA, ultimately allowing for use of the reagent in ribosomal translation.
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Biomimetic Aminoacylation: Investigating Detection of Acylation and the Effect of α-Amino ProtectionAndrusiak, Tara 15 December 2009 (has links)
Direct synthesis of aminoacyl-tRNA occurs using α-N-tBoc-protected aminoacyl phosphates and lanthanum salts. Deprotection of aminoacyl tRNA is essential prior to translation; however, the conditions of tBoc deprotection causes tRNA degradation. It was found that α-N-pentenoyl-protected aminoacyl phosphates, deprotected under mild conditions, are effectively used in lanthanum-mediated acylation of tRNA analogs. This provides an alternative route for aminoacyl-tRNA synthesis that maintains tRNA structure. Also, it was determined that α-N-deprotection of aminoacyl phosphates prior to aminoacylation still produces aminoacylated tRNA analogs. This establishes that acyl phosphates activate amino acids without inducing self-condensation, presumably due to electrostatic repulsion. Direct quantification of lanthanum-mediated tRNA aminoacylation was additionally undertaken utilizing a radiolabelled tRNA assay. From this, it was shown that lanthanum-mediated acylation does not promote deacylation and degradation of tRNA. These results have provided insight into lanthanum-mediated acylation of tRNA, ultimately allowing for use of the reagent in ribosomal translation.
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Biomimetic Aminoacylation: Optimization of Reaction ConditionsBunn, Shannon Elizabeth 05 January 2010 (has links)
Synthesizing proteins containing unnatural amino acids inserted at specific positions within the protein sequence has been a longstanding goal of biological chemists. This poses unique challenges, as aminoacyl tRNA synthetases, the enzymes responsible for protein synthesis, are highly specific. To overcome this, a lanthanum-catalyzed, biomimetic tRNA aminoacylation method has been developed(1). However, due to unproductive lanthanum coordination of ethyl phosphate, a reaction byproduct, a full equivalent of lanthanum must be added to each reaction. This may threaten the integrity of tRNA, as lanthanides are known to catalyze the hydrolysis of RNA (2, 3). Using uridine as a simplified tRNA mimic, magnesium, which is known to coordinate strongly with phosphate ions, has been utilized to optimize this reaction and increase the selectivity of lanthanum towards esterification. In the presence of magnesium, ester yield is substantially increased. In addition to this, optimal pH and buffer reaction conditions were determined.
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Lanthanum-mediated Biomimetic AminoacylationHer, Sohyoung 15 November 2013 (has links)
Methods are being developed to produce “designer proteins” from unnatural amino acids that are added into specific locations by the ribosome using an altered mRNA. To date, over seventy unnatural amino acids have been incorporated at specific sites in proteins by in vitro biosynthetic methods using chemically acylated-tRNAs and in vivo protein mutagenesis based on orthogonal tRNA/aminoacyl-tRNA synthetase pairs.
Lanthanum-mediated aminoacylation of cis-diols provides a general and selective method for the one-step preparation of aminoacyl-tRNA. The nature of this biomimetic process was studied for the reaction of ribonucleosides and nucleotides with N-t-Boc-protected aminoacyl ethyl phosphates. Successful aminoacylation was also achieved with unprotected aminoacyl ethyl phosphates. This method was extended for the aminoacylation of tRNA and analyzed by reversed-phased HPLC and MALDI-MS. These results will provide an insight to the ultimate goal of lanthanum-mediated direct acylation of tRNA and its applications in in vitro site-specific incorporation of unnatural amino acids.
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Lanthanum-mediated Biomimetic AminoacylationHer, Sohyoung 15 November 2013 (has links)
Methods are being developed to produce “designer proteins” from unnatural amino acids that are added into specific locations by the ribosome using an altered mRNA. To date, over seventy unnatural amino acids have been incorporated at specific sites in proteins by in vitro biosynthetic methods using chemically acylated-tRNAs and in vivo protein mutagenesis based on orthogonal tRNA/aminoacyl-tRNA synthetase pairs.
Lanthanum-mediated aminoacylation of cis-diols provides a general and selective method for the one-step preparation of aminoacyl-tRNA. The nature of this biomimetic process was studied for the reaction of ribonucleosides and nucleotides with N-t-Boc-protected aminoacyl ethyl phosphates. Successful aminoacylation was also achieved with unprotected aminoacyl ethyl phosphates. This method was extended for the aminoacylation of tRNA and analyzed by reversed-phased HPLC and MALDI-MS. These results will provide an insight to the ultimate goal of lanthanum-mediated direct acylation of tRNA and its applications in in vitro site-specific incorporation of unnatural amino acids.
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Unraveling the Mystery for the Coexistence of Two Forms of Arginyl-tRNA Synthetase in Mammalian CellsKyriacou, Sophia Vasou 22 September 2008 (has links)
The aminoacyl-tRNA synthetases are among the major protein components in the translation machinery. These essential proteins are responsible for charging their cognate tRNAs with the correct amino acid. Mammalian arginyl-tRNA synthetase (ArgRS), unlike all other eukaryotic aminoacyl-tRNA synthetases, is unique due to the coexistence of two structurally distinct forms of the same enzyme within the same cell: a complexed (or high molecular weight) form that is part of the multi-synthetase complex, and a free (or low molecular weight) form. Until now, not much information is known as to why the cell would synthesize and utilize two different forms of the same enzyme. Do the two forms of ArgRS perform similar or different biological functions? The main hypothesis that was originally proposed is that only the complexed form of ArgRS plays a crucial role in protein synthesis, while the free form of this enzyme participates in the ubiquitination pathway by tagging proteins with acidic NH2-termini (destined for degradation) with an arginine residue on their NH2-terminal end which will serve as a signal for ubiquitin-mediated destruction. Based on my studies, the data indicate that the high molecular weight form of ArgRS, which is present exclusively as an integral component of the multisynthetase complex, is essential for normal protein synthesis and growth of CHO cells even when low molecular weight, free ArgRS is present and Arg-tRNA continues to be synthesized at close to wild type levels. Based on these observations, we can conclude that Arg-tRNA generated by the synthetase complex is a more efficient precursor for protein synthesis than Arg-tRNA generated by free ArgRS, exactly as would be predicted by the channeling model for mammalian translation. No phenotype has been determined for cells expressing only the complexed form of ArgRS, and no direct interaction has been observed between ArgRS and arginyl-tRNA-protein transferase (ATE). Based on this information, we suggest that the function(s) of the free form of ArgRS is either not necessary or is performed by the complexed form when the free form is missing.
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Biomimetic AminoacylationTzvetkova, Svetlana 01 August 2008 (has links)
Abstract
“Biomimetic Aminoacylation”
Svetlana K. Tzvetkova
Doctor of Philosophy, 2008
Graduate Department of Chemistry
University of Toronto
The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA.
Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported.
We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular.
The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.
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Biomimetic AminoacylationTzvetkova, Svetlana 01 August 2008 (has links)
Abstract
“Biomimetic Aminoacylation”
Svetlana K. Tzvetkova
Doctor of Philosophy, 2008
Graduate Department of Chemistry
University of Toronto
The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA.
Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported.
We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular.
The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.
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