The use of 5′-pbospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA

Robertson, Stephanie A.; Noren, Christopher J.; Anthony-Cahill, Spencer J.; Griffith, Michael; Schultz, Peter G.

doi:10.1093/nar/17.23.9649

Cited by 127 publications

(112 citation statements)

References 14 publications

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“…Preparation of NVOC-Protected Aminoacyl-pdCpA. NVOC-protected aminoacyl-pdCpAs were prepared as reported previously by reacting the dinucleotide pdCpA with N-protected, carboxylic acid-activated, amino acids (54)(55)(56).…”

Section: Methodsmentioning

confidence: 99%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

106

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Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe singlemolecule chemistry, and in the construction of nano-and microdevices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼10 4 times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and highresolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.alpha-hemolysin | single molecule | stochastic sensing | structure | unnatural amino acid M any research groups have used protein engineering to obtain enzymes and antibodies with new properties suited for specific tasks (1-6). Fewer groups have taken on the difficult problem of engineering membrane proteins (7). We have engineered the α-hemolysin protein pore, mindful of several potential applications in biotechnology, including its ability to act as a detector in stochastic sensing (8) and ultrarapid DNA sequencing (9), to serve as a nanoreactor for the observation of singlemolecule chemistry (10) and to act as a component for the construction of nano-and microdevices (11).An important breakthrough in this area, which enabled the stochastic sensing of organic molecules including the detection of DNA bases in the form of nucleoside monophosphates (12, 13), was the discovery of internal molecular adapters, a form of noncovalent protein modification (14). Most useful have been cyclodextrin (CD) adapters, which have until now been used in the absence of detailed structural information about how they work. The present paper is a definitive investigation, which provides such information through the application of a wide variety of technical approaches: single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and x-ray crystallography. The studies employing mutagenesis show that the striking interactions seen in the crystal structures also occur in individual pores in lipid bilayers.We reveal that the tight-binding αHL mutants (15) M113N 7 and M113F 7 bind βCD in different orientations within the heptameric pore. In the case of M113N 7 , the top (primary hydroxyls) of the CD ring faces the trans entrance of the pore. In the case of M113F 7 , the bottom (secondary hydroxyls) of the CD ring faces the trans entrance, while the top of the ring is bonded to the pore through remarkable CH-π interactions. Another tight-binding mutant, M113V 7 , can bind the CD in both orientations. These results illustrate the exquisite level of engineering that can be achieved with protein nanopores, which is, for example, far beyond what is possible with solid-state pores. The work also provides information valuable for the design of ...

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Section: Methodsmentioning

confidence: 99%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

106

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“…ACh chloride and yeast inorganic pyrophosphatase were purchased from Sigma-Aldrich. dCA and 6-nitroveratryloxycarbonyl protected dCA-W was prepared as reported in Robertson et al (1989) and Zhong et al (1998). tRNA gene preparation and tRNA transcription THG73, YFFS CCCG , and YFaFS ACCC subcloned in the pUC19 vector were previously made (Saks et al 1996;Rodriguez et al 2006). Genes for ENAS (sequence from Cload et al 1996) with flanking EcoRI and BamHI overhangs were phosphorylated using the Kinase Max kit, annealed, and ligated with T4 DNA ligase into EcoRI and BamHI linearized pUC19 vector as described (Nowak et al 1998).…”

Section: Methodsmentioning

confidence: 99%

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Rodriguez¹,

Lester²,

Dougherty³

2007

RNA

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The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.

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“…Applying the same conditions to the lanthanum ion-promoted aminoacylation of AMP increased the yield from around 30% to 40%. For aminoacylation of dCA, a dinucleotide used by Robertson et al (1989) in the chemical aminoacylation of tRNA, the combined yields of 2′ and 3′ esters doubled from 6.8% to 15% after 12 h of reaction. With PheEtP at its saturating concentration, the reaction produced the desired esters at a 40% yield after 12 h at 4°C (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Increased efficiency in biomimetic Lewis acid–base pair catalyzed monoacylation of diols by acyl phosphate monoesters

Kluger

2017

FACETS

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Acyl phosphate monoesters are biomimetic acylation reagents that require coordination to metal ions to react with cis-diol substrates in water. With lanthanide catalysts, outcomes are compromised by (1) the competitive lanthanide-promoted hydrolysis of the acyl phosphate reagents as well as by (2) the high affinity of lanthanum ions for the phosphate monoester by-product. Based on analysis of the mechanism of the process, optimizing reaction conditions can selectively inhibit the lanthanum-promoted hydrolysis of acyl phosphate monoesters. Furthermore, using zinc salts and lead salts in place of lanthanides enhances the reactivity of the reactants and causes less complexation of the metal ion with the by-products.

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The use of 5′-pbospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA

Cited by 127 publications

References 14 publications

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Increased efficiency in biomimetic Lewis acid–base pair catalyzed monoacylation of diols by acyl phosphate monoesters

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