Measure of carbon and nitrogen stable isotope ratios in cultured cells.

We report the measurement of the natural isotope ratios of nitrogen and carbon in subcellular volumes of individual cells among a population of cultured cells using a multi-isotope imaging mass spectrometer (MIMS), [MIMS is the prototype of the NanoSIMS 50, Cameca, France.] We also measured the nitrogen and carbon isotope ratio in cells after they had been cultured in media enriched with the amino acid glycine labeled with either 13C or 15N. The results demonstrate that 13C/12C and 15N/14N isotope ratios can be measured directly on a subcellular scale. This opens the way for the use of stable isotopes, in particular 15N, as labels to measure the intracellular turnover of biomolecules. Such a capability should help resolve a wide range of biomedical problems.

Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods.

The solution conformation of the cyclic peptide J324 (cyclo0,6-[Lys0,Glu6,D-Phe7]BK), an antagonist targeted at the bradykinin (BK) B2 receptor, has been investigated using experimental and theoretical methods. In order to gain insight into the structural requirements essential for BK antagonism, we carried out molecular dynamics (MD) simulations using simulated annealing as the sampling protocol. Following a free MD simulation we performed simulations using nuclear Overhauser enhancement (NOE) distance constraints determined by NMR experiments. The low-energy structures obtained were compared with each other, grouped into families and analyzed with respect to the presence of secondary structural elements in their backbone. We also introduced new ways of plotting structural data for a more comprehensive analysis of large conformational sets. Finally, the relationship between characteristic backbone conformations and the spatial arrangement of specific pharmacophore centers was investigated.

Biochemical and biophysical characterization of the trimerization domain from the heat shock transcription factor.

Previously, we had characterized a 91 amino acid fragment of the heat shock transcription factor from the yeast Kluyveromyces lactis and had shown it to be highly alpha-helical and sufficient for formation of homotrimers [Peteranderl, R., and Nelson, H. C. M. (1992) Biochemistry 31, 12272-12276]. Based on those data, as well as the presence of hydrophobic heptad repeats, we postulated that the trimerization domain contains a three-stranded coiled-coil and that it might resemble the trimerization domain found in influenza hemagglutinin. Here, we further characterize the trimerization domain and show that the minimal domain needs 71 residues to remain trimeric and highly alpha-helical. 19F NMR spectroscopy suggests that the structure contains three parallel strands that are in register along the long axis of the coiled-coil. Electron paramagnetic resonance spectroscopy studies show that the C-termini of the subunits are in close proximity; this is in contrast to the topology of the hemaglutinin trimerization domain where the C-termini form buttressing helices. Analytical ultracentrifugation also confirms that the structure is elongated and unlikely to have buttressing helices. Additional experiments suggest that the trimerization domain has at least two subdomains. The first subdomain has the potential to form trimers independently, though not as stably as the complete domain. The second subdomain is quite helical, forms large oligomers, and appears to provide stability to the complete domain. Our current model for the heat shock transcription factor trimerization domain is a highly elongated coiled-coil structure, with a potential break in the coiled-coil region located between the two subdomains.

Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.

The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).

Induction and Regeneration of Autoplasts from Clostridium thermohydrosulfuricum JW102 and Thermoanaerobacter ethanolicus JW200.

Autolysis was induced to form stable, cell wall-free cells of Clostridium thermohydrosulfuricum JW102 and Thermoanaerobacter ethanolicus JW200, using a complex medium containing glycine (0.4% wt/vol) and/or sucrose or glycerol (10% wt/vol) at an optimum temperature of 64 degrees C. Autoplasts of both bacteria were grown as L-phase colonies on solid medium; more than 50% of these colonies regenerated to the walled form during prolonged incubation. The removal of the cell wall was confirmed by electron microscopy.

Trimerization of the heat shock transcription factor by a triple-stranded alpha-helical coiled-coil.

We have isolated and characterized a 91 amino acid fragment of the heat shock transcription factor from both Saccharomyces cerevisiae and Kluyveromyces lactis. The two protein fragments behave similarly: they form homotrimers, as indicated by sedimentation equilibrium and cross-linking, and contain approximately 80% alpha-helix, as indicated by circular dichroism. Sedimentation velocity and diffusion coefficients indicate that they have an elongated, nonspherical shape. We conclude the following: these fragments contain a domain which forms a trimer via a triple-stranded alpha-helical coiled-coil, similar to that found in influenza hemagglutinin.

Stability of antibiotics under growth conditions for thermophilic anaerobes.

It was shown that the inhibitory effect of kanamycin and streptomycin in a growing culture of Clostridium thermohydrosulfuricum JW 102 is of limited duration. To screen a large number of antibiotics, their stability during incubation under the growth conditions of thermophilic clostridia was determined at 72 and 50 degrees C by using a 0.2% yeast extract-amended prereduced mineral medium with a pH of 7.3 or 5.0. Half-lives were determined in a modified MIC test with Escherichia coli, Staphylococcus aureus, and Bacillus megaterium as indicator strains. All compounds tested were similar at the two temperatures or more stable at 50 than at 72 degrees C. The half-life (t1/2) at pH 7.3 and 72 degrees C ranged from 3.3 h (k = 7.26 day-1, where k [degradation constant] = 1/t1/2) for ampicillin to no detectable loss of activity for kanamycin, neomycin, and other antibiotics. Apparently some compounds (e.g., lasalocid and neomycin) became more potent during incubation (k greater than 0). A change to pH 5.0 caused some compounds to become more labile (e.g., kanamycin) and others (e.g., streptomycin) to become more stable than at pH 7.3.