<i>p</i>-Thiophenylalanine-Induced DNA Cleavage and Religation Activity of a Modified Vaccinia Topoisomerase IB

Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5′(C/T)­CCTTp↓ sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (kcl = 1 × 10–4 s–1) was 30 times lower than for the wild-type enzyme (kcl = 3 × 10–3 s–1). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O–P and S–P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg2+ and Mn2+ to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg2+ and Mn2+ increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion–heteroatom interaction is required for catalysis

Stereoselective Synthesis of the Atropisomers of Myristinin B/C

The first stereoselective synthesis of a potent DNA damaging agent, (−)-myristinin B/C, has been accomplished. This efficient synthesis allowed for unambiguous confirmation of the structure and absolute stereochemistry of the atropisomeric natural product. The antipode, (+)-myristinin B/C, was also synthesized, providing ample material for biological evaluation of both enantiomers

Selective Detection of Dengue Virus Serotypes Using Tandem Toehold-Mediated Displacement Reactions

Dengue virus (DENV) is the most common human arboviral infection worldwide and can present with severe clinical manifestations. Timely DENV detection improves clinical outcomes, and identification of the DENV serotype (DENV-1–4) may provide beneficial epidemiologic data to inform the initiation of control measures. Here, DENV RNA-triggered, enzyme-free tandem toehold-mediated displacement reactions were developed to identify and serotype DENV in RNA controls and contrived samples through the amplification of a fluorescent signal detected by the use of a fluorescent scanner and a confocal microscope. Each DENV serotype was detected selectively using both imaging methods. In addition, a 384-well plate was used to prepare an array for diagnosis of the four DENV RNA serotypes from contrived clinical samples. The four serotypes of dengue virus were detected using novel enzyme-free amplification reactions, which are more facile than amplification using reverse transcriptase PCR

Efficient Asymmetric Synthesis of Tryptophan Analogues Having Useful Photophysical Properties

Two new fluorescent probes of protein structure and dynamics have been prepared by concise asymmetric syntheses using the Schöllkopf chiral auxiliary. The site-specific incorporation of one probe into dihydrofolate reductase is reported. The utility of these tryptophan derivatives lies in their absorption and emission maxima which differ from those of tryptophan, as well as in their large Stokes shifts and high molar absorptivities

A New Strategy for the Synthesis of Bisaminoacylated tRNAs

Tandemly activated tRNAs participate effectively in protein synthesis and exhibit superior chemical and biochemical stability compared to the more commonly used singly aminoacylated tRNAs. While several bisaminoacylated tRNAs have been prepared via the T4 RNA ligase-mediated condensation of bisaminoacylated pdCpAs and abbreviated tRNA transcripts (tRNA-COH), the bisaminoacylated pdCpAs are difficult to prepare when using bulky amino acids. Described herein is a new strategy for preparing bisaminoacylated tRNAs, applicable even for bulky amino acids

<i>In Cellulo</i> Synthesis of Proteins Containing a Fluorescent Oxazole Amino Acid

Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved α-l-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpuromycin analogue, were employed to incorporate dipeptides and dipeptidomimetics. Presently, we report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asymmetric center or α-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (PylRS). Initially, a plasmid encoding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium containing oxazole 2 resulted in the elaboration of RRM1 containing the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo containing oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homologue of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo

Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription

The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases

Two Pyrenylalanines in Dihydrofolate Reductase Form an Excimer Enabling the Study of Protein Dynamics

Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl-tRNACUA. Excimer formation (λex 342 nm; λem 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation

Alteration of Transcriptional Regulator Rob <i>In Vivo</i>: Enhancement of Promoter DNA Binding and Antibiotic Resistance in the Presence of Nucleobase Amino Acids

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson–Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson–Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the β-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues

Activation of d‑Asparagine and d‑Glutamine Derivatives Using the Mitsunobu Reaction

Seven d-amino acid derivatives having reactive side chains have been activated to afford their respective 3,5-dinitrobenzyl esters using the Mitsunobu reaction. This esterification was found to be difficult using traditional methods involving 3,5-dinitrobenzyl chloride under alkaline conditions. The conversion of a tRNA to the respective d-glutaminyl-tRNA using d-glutamine 3,5-dinitrobenzyl ester was catalyzed by a flexizyme, followed by purification to remove all the unacylated tRNAs and other byproducts. Both d- and l-glutamine were incorporated from their aminoacyl-tRNAs into a model peptide structurally related to IFN-β