[15] Biosynthetic method for introducing unnatural amino acids site-specifically into proteins

Ellman, Jon; Mendel, David; Anthony-Cahill, Spencer J.; Noren, Christopher J.; Schultz, Peter G.

doi:10.1016/0076-6879(91)02017-4

Cited by 194 publications

(177 citation statements)

References 23 publications

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“…T4 polynucleotide kinase was prepared as described by Cameron and Uhlenbeck (30). T7 RNA polymerase was prepared using Escherichia coli BL21/DE3, containing the plasmid pAR1219 (ampicillin resistance) in which the gene for T7 RNA polymerase is under the transcription control of the lac promoter (31). All enzymes were stored at -20°C in glycerol solutions.…”

Section: Methodsmentioning

confidence: 99%

Probing the role of loop 2 in Ras function with unnatural amino acids.

Chung

Benson²,

Cornish³

et al. 1993

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

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The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crystal structures have revealed significant conformational differences in this region between the GTP-and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interaction with GTPase-activating protein (GAP), suggesting that this region may contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, . A Pro-34--methanoproline mutant, in which residue 34 is locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Be (Ile-36 --norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.Mammalian proteins encoded by the ras genes function as molecular switches in the signaling events associated with cell growth and differentiation (1-4). The chemical basis for the regulation involves cycling of the protein between the inactive (off) GDP-bound state and the active (on) GTPbound state. The low intrinsic GTPase activity ofcellular Ras protein is increased by interaction with the cytosolic protein GTPase-activating protein (GAP), a possible effector or negative regulator of Ras. In addition, GDP-GTP exchange factors (5-8), which have recently been identified as possible positive regulators, enhance the dissociation ofGDP from the protein and facilitate rebinding of GTP. Point mutations that result in a modest decrease in the intrinsic GTPase activity of Ras or GAP (9-12)-stimulated GTPase activity are associated with a large number of human cancers.The YDPT sequence motif (residues 32-35 in loop 2 ofRas) is conserved throughout the Ras protein family. X-ray crystal structures (13-20) of the GDP-, guanosine 5'-[,8,y-methyleneltriphosphate-, and guanosine 5'-[P,lyimido]triphosphate-bound forms of Ras have shown that the loop 2 region undergoes significant conformational changes upon hydrolysis ofGTP. The 3-hydroxyl group ofThr-35, which associates with Mg2+ in the GTP-Ras complex, moves out toward solvent in the GDP complex. In addition, the side chain of Ile-36, which is solvent-exposed in the GTP complex, is positioned closer to the protein surface in the GDP complex. This flexibility has been proposed to be a functionally important feature of Ras and perhaps in the GTPase family in general. in this region that block neoplastic transformation also prevent interaction ofRas with GAP. WeThe publication costs of this article were defrayed in p...

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Section: Methodsmentioning

confidence: 99%

Probing the role of loop 2 in Ras function with unnatural amino acids.

Chung

Benson²,

Cornish³

et al. 1993

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

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“…1 Preparation of CCA-Phe-X-Biotin (Scheme 2)-Oligonucleotides were handled in diethyl pyrocarbonate-treated sterile water. The procedure of Ellman et al (13) was modified. The CCA trinucleotide was converted to the tetra-n-butylammonium salt by passing an aqueous solution of trinucleotide through a 5-g column of Bio-Rex 70 resin (prepared from the sodium form by washing sequentially with 70% ethanol-water, water, 0.1 N aqueous sodium hydroxide, water, 0.1 N aqueous hydrochloric acid, water until neutral, 0.1 N tetra-n-butylammonium hydroxide, and water until neutral).…”

Section: Preparation Of N-(6-((biotinoyl)amino)hexanoyl)-l-phenylalanmentioning

confidence: 99%

Oxazolidinones Mechanism of Action: Inhibition of the First Peptide Bond Formation

Patel

Yan

Hobbs

et al. 2001

Journal of Biological Chemistry

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Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steadystate kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA i Met to the ribosomal peptidyltransferase Psite, which is vacant only prior to the formation of the first peptide bond.

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“…While activity screens make it possible to obtain large data sets made up of sequences that produce functional and presumably stable proteins (Bowie et al, 1990), overexpression and purification of selected protein variants can provide enough material for extensive physical studies. However, mutagenesis techniques are limited to the set of 20 natural amino acids, although approaches to circumnavigate this limitation are available (Ellman et al, 1991;Mendel et al, 1992).…”

mentioning

confidence: 99%

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques

Wynn

Richards

1993

Protein Science

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We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesiskhemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols. These mutants all show native-like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmC1],,2, and the free energy of unfolding at zero denaturant concentration, AG(H20), give inverse orders of stability. This effect is due to changes in m, the dependence of AGinfolding on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.

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[15] Biosynthetic method for introducing unnatural amino acids site-specifically into proteins

Cited by 194 publications

References 23 publications

Probing the role of loop 2 in Ras function with unnatural amino acids.

Probing the role of loop 2 in Ras function with unnatural amino acids.

Oxazolidinones Mechanism of Action: Inhibition of the First Peptide Bond Formation

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques

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