John W. Trauger

Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes

This chapter describes three complementary footprinting methods and the protocols used for analysis of polyamide : DNA complexes: (1) MPE–Fe(II) footprinting, (2) affinity cleavage, and (3) quantitative DNase I footprint titration. Footprinting with methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] is used to identify high-affinity polyamide-binding sites to near nucleotide resolution. Affinity cleavage is used to determine the orientation of the bound polyamide in the minor groove of DNA. Quantitative DNase I footprinting is used to determine equilibrium association constants (K_a) for polyamide–DNA complexes at previously identified match and mismatch sites. A pivotal step in the discovery—evaluation process of new polyamide motifs is the characterization of the affinity and specificity of next-generation molecules following the design-sythesis phase. In the design phase, it is often the case that the sequence preference, as well as the energetics of any new molecule binding at each potential site, is not perfectly understood and it is crucial to scan “libraries” of many potential DNA-binding sites in order to identify true high-affinity binding sites.

Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.

Inhibition of Ets-1 DNA Binding and Ternary Complex Formation between Ets-1, NF-kappa B, and DNA by a Designed DNA-binding Ligand

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-κB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1·NF-κB·DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-κB also inhibits NF-κB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Chain Termination Steps in Nonribosomal Peptide Synthetase Assembly Lines : Directed Acyl-S-Enzyme Breakdown in Antibiotic and Siderophore Biosynthesis

A large number of therapeutically useful natural peptides areproduced nonribosomally by assembly line enzymology, involv-ing multidomain and multimodular catalysts that activate andassemble constituent amino acid monomers into oligopeptides.The peptide chains released (Scheme 1) can be final productssuch as cyclosporin (1)

HST far-ultraviolet imaging of Jupiter during the impacts of comet Shoemaker-Levy 9

Hubble Space Telescope far-ultraviolet images of Jupiter during the Shoemaker-Levy 9 impacts show the impact regions darkening over the 2 to 3 hours after the impact, becoming darker and more extended than at longer wavelengths, which indicates that ultraviolet-absorbing gases or aerosols are more extended, more absorbing, and at higher altitudes than the absorbers of visible light. Transient auroral emissions were observed near the magnetic conjugate point of the K impact site just after that impact. The global auroral activity was fainter than average during the impacts, and a variable auroral emission feature was observed inside the southern auroral oval preceding the impacts of fragments Q1 and Q2.

Supernova Remnants and the Interstellar Medium of M83: Imaging and Photometry with the Wide Field Camera 3 on the Hubble Space Telescope

We present Wide Field Camera 3 images taken with the Hubble Space Telescope within a single field in the southern grand design star-forming galaxy M83. Based on their size, morphology, and photometry in continuum-subtracted Hα, [S II], Hβ, [O III], and [O II] filters, we have identified 60 supernova remnant (SNR) candidates, as well as a handful of young ejecta-dominated candidates. A catalog of these remnants, their sizes and, where possible, their Hα fluxes are given. Radiative ages and pre-shock densities are derived from those SNRs that have good photometry. The ages lie in the range 2.62 < log (τrad/yr) < 5.0, and the pre-shock densities at the blast wave range over 0.56 < n 0/cm-3 < 1680. Two populations of SNRs have been discovered. These divide into a nuclear and spiral arm group and an inter-arm population. We infer an arm to inter-arm density contrast of 4. The surface flux in diffuse X-rays is correlated with the inferred pre-shock density, indicating that the warm interstellar medium (ISM) is pressurized by the hot X-ray plasma. We also find that the ISM in the nuclear region of M83 is characterized by a very high porosity and pressure, and infer an SNR rate of 1 per 70-150 yr for the nuclear (R < 300 pc) region. On the basis of the number of SNRs detected and their radiative ages, we infer that the lower mass of Type II SNe in M83 is M min = 16+7 –5 M ☉. Finally, we give evidence for the likely detection of the remnant of the historical supernova, SN1968L.

Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells.

Rho-kinase II (ROCK-II) is a serine/threonine kinase that is involved in regulation of smooth muscle contraction and has been shown to contribute to the early stages of axon formation in neurons and the regulation of the neuronal cytoskeleton. Much of what is known about Rho-kinase function comes from cell-biological studies, whereas a paucity of biochemical characterization exists for the enzyme. In an effort to characterize ROCK-II biochemically we have cloned a truncated form of human ROCK-II comprising amino acids 1-543 and overexpressed it in Sf-21 cells. Utilizing the Sf-21/baculovirus expression system we isolated milligram quantities of ROCK-II (1-543) and purified the enzyme to near homogeneity. Optimal expression conditions revealed that infection of Sf-21 cells at a multiplicity of infection of 10 for 72h yielded maximal protein expression. Expression of ROCK-II (1-543) as an N-terminal Flag fusion protein allowed a single-step purification yielding greater than 90% homogeneous protein as assessed by SDS-PAGE. Enzyme activity was linear over a range of enzyme concentrations and times. Capture of phosphorylated, biotinylated peptides on streptavidin membrane allowed assessment of peptide substrate preference and measurement of steady-state rate constants. The data indicated that an 11-mer peptide containing Ser235/Ser236 of the S6 ribosomal protein and a 12-mer peptide containing Thr508 of LIM kinase were preferred substrates for ROCK-II (1-543). Finally, staurosporine had an IC(50) value 215-fold more potent than that of the ROCK inhibitor Y-27632. Collectively these data lay the foundation for the beginning of a biochemical characterization for this enzyme and provide methodology for more detailed biochemical, biophysical, and kinetic analysis.

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins.

Cooperative hairpin dimers for recognition of DNA by pyrrole-imidazole polyamides

A doubling of the length of binding site for the same size of ligand is achieved by the title compound by formation of a cooperative hairpin dimer on binding to DNA (depicted schematically below). The binding affinity and selectivity are unaffected by this new binding pattern. Circles represent heterocyclic rings, and diamonds and curved lines represent β-alanine and (R)-2,4-diaminobutyric acid residues, respectively.

Pupil mapping exoplanet coronagraphic observer (PECO)

The Pupil mapping Exoplanet Coronagraphic Observer (PECO) mission concept is a 1.4-m telescope aimed at imaging and characterizing extra-solar planetary systems at optical wavelengths. The coronagraphic method employed, Phase-Induced Amplitude Apodization or PIAA (a.k.a. pupil mapping) can deliver 1e-10 contrast at 2 lambda/D and uses almost all the starlight that passes through the aperture to maintain higher throughput and higher angular resolution than any other coronagraph or nuller, making PECO the theoretically most efficient existing approach for imaging extra-solar planetary systems. PECOs instrument also incorporates deformable mirrors for high accuracy wavefront control. Our studies show that a probe-scale PECO mission with 1.4 m aperture is extremely powerful, with the capability of imaging at spectral resolution R≈∠15 the habitable zones of already known F, G, K stars with sensitivity sufficient to detect planets down to Earth size, and to map dust clouds down to a fraction of our zodiacal cloud dust brightness. PECO will acquire narrow field images simultaneously in 10 to 20 spectral bands covering wavelengths from 0.4 to 1.0 μm and will utilize all available photons for maximum wavefront sensing and imaging/spectroscopy sensitivity. This approach is well suited for low-resolution spectral characterization of both planets and dust clouds with a moderately sized telescope. We also report on recent results obtained with the laboratory prototype of a coronagraphic low order wavefront sensor (CLOWFS) for PIAA coronagraph. The CLOWFS is a key part of PECOs design and will enable high contrast at the very small PECO inner working angle.