PubMed Health⌕ Search

Biomedical subjects

J Langowski

Publications and source records attributed to J Langowski.

16 recordsLinked to original sources
PubMed1992

Preparation of DNA topoisomers by RP-18 high-performance liquid chromatography.

A method for the separation of superhelical DNA on the basis of superhelical density by reverse-phase HPLC on RP-18 columns is described. The technique can be used to prepare superhelical DNA in milligram amounts and narrow topoisomer distributions in 0.1 mg amounts. We show example separations of the plasmids pUC18 (2687 bp) and pi AN13 (895 bp). While the best separation for pUC18 yields topoisomer distributions of two or three major components, the small plasmid can be separated into single topoisomer fractions. The basis of the separation is probably an interaction of partially opened bases with the hydrophobic column matrix. This hypothesis is supported by the elution behavior of DNA fragments on this column: DNA fragments with sticky ends, even at a length of several hundred base pairs, elute at much higher methanol concentrations than blunt-ended fragments.

Chromatography, High Pressure Liquid↗
PubMed1992

Pre-nucleation crystallization studies on aminoacyl-tRNA synthetases by dynamic light-scattering.

Dynamic light-scattering (DLS) studies on solutions of proteins approaching their precipitation point were made with asparaginyl- (NRSEC), leucyl- (LRSEC) and valyl- (VRSEC) tRNA synthetases from Escherichia coli. The three aminoacyl-tRNA synthetases have not been crystallized previously. As a control system, we used E. coli polypeptide elongation factor Tu (EF-Tu). Apart from the different proteins used here, the methods we employed differed from previous studies in that (1) instead of making a series of measurements on individual samples at various concentrations, the protein solutions were titrated with the precipitants, and (2) the results of the light-scattering measurements were analysed by a new maximum entropy procedure that calculates a particle size distribution in a highly reproducible way. The particle size distributions of protein solutions titrated with precipitants showed two major peaks in most cases. For both peaks, relative areas and mean diffusion coefficients were determined. The diffusion constants were corrected for the viscosity of the solutions. From comparing the results on the proteins known to crystallize (EF-Tu) with the amorphously precipitating systems (LRSEC, NRSEC) we find two necessary, but not sufficient, conditions for the formation of crystals: the diffusion coefficient of the monomer peak stays constant until very close to the precipitation point; the percentage of large aggregates stays small (less than 10% of the scattered light intensity) during the titration. For VRSEC, both ammonium sulphate and sodium citrate showed a low percentage of large aggregates and a constant diffusion coefficient of the main (protein monomer) peak below the precipitation point. This indicates that both would be possible precipitants for the crystallization of this enzyme. Crystallization trials using both these salts were carried out, and although no condition could as yet be found for obtaining crystals with ammonium sulphate solutions, crystals of the enzyme have been obtained with sodium citrate.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Structure and RNA content of the prosomes.

Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

Animals↗
PubMed1992

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.

Bacterial Proteins↗
PubMed1991

Spatial visualization of DNA in solution.

An image analysis method is presented which allows for the reconstruction of the three-dimensional path of filamentous objects from two of their projections. Starting with stereo pairs, this method is used to trace the trajectory of DNA molecules embedded in vitreous ice and leads to a faithful representation of their three-dimensional shape in solution. This computer-aided reconstruction is superior to the subjective three-dimensional impression generated by observation of stereo pairs of micrographs because it enables one to look at the reconstructed molecules from any chosen direction and distance and allows quantitative analysis such as determination of distances, curvature, persistence length, and writhe of DNA molecules in solution.

DNA, Bacterial↗
PubMed1990

Neutron and light-scattering studies of DNA gyrase and its complex with DNA.

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.

Animals↗
PubMed1990

Purification and characterization of wild-type and ts 112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli.

The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously (M. Cuillel, M. Milleville, and J. C. D'Halluin, 1987, Gene 55, 295-301). The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts 112 whose functional mutation occurs in this gene. The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation. Both the wild-type and ts 112 recombinant proteins are produced in E. coli in an insoluble form, but are readily solubilized in urea. They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins. Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter. The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.

Adenoviruses, Human↗
PubMed1989

Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering.

The solution conformation and internal motions of five superhelical DNAs between 2100 and 10200 base-pairs in length have been characterized by dynamic light scattering (DLS). Variations in the diffusion coefficients and rotational relaxation times with molecular weight are both indicative of an anisotropic extended structure of these DNAs; we therefore conclude that under our conditions the interwound superhelical structure prevails. The internal dynamics can be described by a superposition of rotational diffusion and internal relaxation. The latter process is characterized by the internal diffusion of persistence length size segments within the DNA chain and faster bending motions within these segments.

Chemical Phenomena↗
PubMed1988

An elongated model of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex derived from neutron scattering and hydrodynamic measurements.

The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700 +/- 10,000 and 86,700 +/- 9000 daltons from these two methods respectively. The observed match point of 54.4% D2O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. At high neutron scattering contrast radius of gyration of 42.3 +/- 2 A was found for the 7S particle. In addition a diffusion coefficient of 4.4 x 10(-11) [m2 s-1] and a sedimentation coefficient of 6.2S were determined. The hydrodynamic radius obtained for the 7S particle is 48 +/- 5 A. A simple elongated cylindrical model with dimensions of 140 A length and 59 A diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 A in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.

Animals↗
PubMed1987

Salt effects on internal motions of superhelical and linear pUC8 DNA. Dynamic light scattering studies.

The plasmid pUC8 (2717 bp) has been studied in its native superhelical and Eco RI-linearized forms by dynamic light scattering at NaCl concentrations from 1.1 mM to 1 M. The data were analyzed using the biexponential model for the dynamic structure factor described by us in a previous paper (J. Langowski, U. Giesen and C. Lehmann, Biophys. Chem. 25 (1986) 191). As before, we could identify two decay components corresponding to the center-of-mass diffusion and to internal motions of the DNA, where the fast component could be identified as a rotational diffusion contribution in the case for superhelical, but not for linear DNA. We found that the conformation of superhelical pUC8 is not affected by changing the ionic strength, while the amplitude of the internal relaxation increases approx. 2-fold when [NaCl] is raised from 1.1 mM to 1 M. The linearized DNA shows an increase of the diffusion coefficient with ionic strength which is, however, not quite as pronounced as that found by others (Z. Kam, N. Borochov and H. Eisenberg, Biopolymers 20 (1981) 2671), and, together with the unchanged conformation of the superhelical DNA, suggests a persistence length which is not strongly dependent on ionic strength. In contrast to the increasing amplitude of internal relaxation for the superhelical DNA, this amplitude remains constant or decreases slightly for linear DNA on going from 1.1 mM to 1 M salt. Our findings are further discussed with respect to possible models of the interwound form of superhelical DNA.

DNA, Superhelical↗
PubMed1986

Dynamics of superhelical DNA studied by photon correlation spectroscopy.

We have conducted photon correlation spectroscopy (PCS) studies on the plasmid pUC8 (2717 bp) in order to elucidate the internal dynamics of this superhelical DNA. We confirm that the first-order autocorrelation function of the scattered light from pUC8 solutions can be separated into two distinct exponential decay components, as first shown by Lewis et al. (R. Lewis, J.H. Huang and P. Pecora, Macromolecules 18 (1985) 944). A thorough analysis of the dependence on scattering vector K of the rates and amplitudes of the two components enables us to assign the slowly relaxing part to the center-of-mass diffusion of the DNA, while the faster component corresponds to rotational, bending and twisting motions of the superhelix. For larger K values the internal motions can be formally expressed in terms of an 'internal diffusion coefficient' Di, whose value of 2.0-2.5 X 10(-11) m2 s-1 is approximately equal to the translational diffusion coefficient predicted for a stiff DNA piece of the persistence length, 65 nm. Comparison of our measured Di values to those predicted from a recent theory of circular worm-like coils (K. Soda, Macromolecules 17 (1984) 2365) shows that the internal motions are faster than the theoretical values. One of the reasons for this discrepancy could be that the theory does not take into account torsional motions, which contribute significantly to the internal dynamics (J.C. Thomas, S.A. Allison, C.J. Appelof and J.M. Schurr, Biophys. Chem. 12 (1980) 177). At low K values, the fast relaxation of superhelical pUC8 is no longer proportional to K2, but reaches a constant value as K approaches zero. This behavior, not seen for the linearized DNA, can be interpreted in terms of rotational diffusion of a flexible rod-like molecule (T. Maeda and S. Fujime, Macromolecules 17 (1984) 2381) and supports an interwound rod-like structure for pUC8 DNA with an average end-to-end distance of 220 nm.

DNA Restriction Enzymes↗
PubMed1985

Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein.

The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.

DNA Helicases↗
PubMed1985

Product analysis of in vitro ribosomal protein synthesis for the assessment of kinetic parameters.

For the characterization of the product distribution of in vitro ribosomal protein synthesis a new method is introduced in which radioactively labeled peptides are separated on a reversed-phase HPLC column and detected on line with a flow radioactivity monitor. Employing this procedure the kinetics of product formation under pre-steady-state conditions were measured under a variety of conditions. These measurements yield the intrinsic monomolecular rate constants for peptidyl transfer (greater than 20 s-1) and translocation (rate limiting for elongation). The usefulness of this technique for accuracy measurements is illustrated.

Carbon Radioisotopes↗
PubMed1978

Common structural features of different viroids: serial arrangement of double helical sections and internal loops.

The thermodynamic parameters of five different highly purified viroid "species" were determined by applying UV-absorption melting analysis and temperature jump methods. Their thermal denaturation proved to be a highly cooperative process with midpoint-temperatures (Tm) between 48.5 and 51 degrees C in 0.01 M sodium cacodylate, 1 mM EDTA, pH 6.8. The values of the apparent reaction enthalpies of the different viroid species range between 3,140 and 3,770 kJ/mol. Although the cooperativity is as high as found in homogeneous RNA double helices the Tm-value of viroid melting is more than 30 degrees C lower than in the homogeneous RNA. In order to explain this deviation, melting curves were simulated for different models of the secondary structure of viroids using literature values of the thermodynamic parameters of nucleic acids. Our calculations show that the following refinement of our earlier model is in complete accordance with the experimental data: In their native conformation viroids exist as an extended rodlike structure characterized by a series of double helical sections and internal loops. In the different viroid species 250-300 nucleotides out of total 350 nucleotides are needed to interprete the thermodynamic behaviour.

Mathematics↗