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Small molecules for riboswitch detection and N-terminal protein modification
Budhathoki, Pradeep
Budhathoki, Pradeep
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[Fort Worth, Tex.] : Texas Christian University,
Date
2015
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Abstract
Riboswitches are metabolite sensing mRNA molecules that regulate gene expression in bacteria, archea, fungi, and plants. These recently characterized molecules specifically bind to an associated metabolite, inducing conformational changes that ultimately lead to negative or positive regulation of the genes associated with the biosynthesis or transport of the bound metabolite. To date, more than 20 distinct classes of riboswitches have been found for different cellular metabolites including various amino acids, sugars, coenzymes, nucleotide bases, and metal ions. In line probing, transcription termination assay and isothermal titration calorimetry have been used to study riboswitches (Chapter 1). However, due to its distinct advantages, fluorescence based approach is useful for probing riboswitches. Based on the three dimensional structure of metabolite-bound riboswitch, fluorescent probes were designed, synthesized and evaluated (chapter 2).^Two fluorescent lysine amide analogs, in which the carboxyl end of lysine was covalently attached to dansyl or NBD groups through an ethylene glycol-based linker showed high binding affinity to the lysine riboswitch in vitro. Protein modification provides an important tool for studying protein structure and function. Due to its low abundance and nucleophilicity, cysteine is useful for selective and site-specific protein modifications (Chapter 3). The N-terminal cysteine residue of proteins can selectively react with pyruvate analogs at pH 7 to form their corresponding 2-methyl-2,4-thiazolidinedicarboxylic acid derivatives (chapter 4). Aminoxy-containing reagents such as methoxylamine can easily reverse this reaction to regenerate the free cysteine residue. Several pyruvate analogs were synthesized with affinity (biotin) and fluorescent (pyrene) labels, which are useful for protein purification and detection, respectively.^Also synthesized were pyruvate analogs with unique chemical handles such as azide and propargyl groups. These functional groups can be used to further modify proteins using bioorthogonal reactions without interfering with native biochemical reactions. All of the prepared pyruvate analogs were successfully tested for the selective and efficient N-terminal modification of the Z-domain as a model protein. The modification was easily removed to regenerate the intact unmodified proteins.
Contents
Subject
Subject(s)
Riboswitches.
Proteins Chemical modification.
Metabolites.
Proteins Chemical modification.
Metabolites.
Research Projects
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Dissertation
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Department
Chemistry and Biochemistry