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Biomedical subjects

D Zirwer

Publications and source records attributed to D Zirwer.

At least 19 recordsLinked to original sources
PubMed1992

Application of dynamic light scattering to studies of protein folding kinetics.

The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally. The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described. The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed. It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g.mol(-1) and concentrations of 1 g.1(-1). Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes. This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.

Kinetics↗
PubMed1992

Streptokinase is a flexible multi-domain protein.

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.

Animals↗
PubMed1991

Acid denatured apo-cytochrome c is a random coil: evidence from small-angle X-ray scattering and dynamic light scattering.

The conformation of a denatured protein has been investigated, since the experimental data on the structure of denatured proteins have been incomplete until now. The Stokes' radius Rs and the radius of gyration Rg of apo-cytochrome c at pH 2.3 have been determined by dynamic light scattering and small-angle X-ray scattering, respectively. The values of these structure parameters, extrapolated to zero protein concentration, are Rs = 3.0 nm and Rg = 4.6 nm. The ratio Rg.Rs-1 is a sensitive indicator of the molecular conformation. The ratio of 1.55 obtained by us is typical for a random-coil polymer. The persistence length--the characteristic of the molecular flexibility--was determined to be a = 1.81 nm. From this results the root-mean-square average end-to-end distance of the molecules [h2] 1/2 = 11.2 nm and the characteristic ratio [h2]/npl2p = 8.43, where np = 104 is the number of amino acid residues and lp the distance between C alpha-atoms. We obtained a second virial coefficient A2 = 8.2.10(-3) mol cm3 g-2. The experimentally determined structure parameters are in approximate agreement with those predicted by Flory and others for an unperturbed, randomly coiled polypeptide. The expansion factor lies between 1.1 and 1.2. In conclusion, we have shown that apo-cytochrome c at pH 2.3 and at low concentrations has the conformation of a perturbed random coil with repulsive potentials between the chain segments.

Animals↗
PubMed1991

Solvent dependence of dimensions of unfolded protein chains.

The radii of gyration of unfolded apo-cytochrome C at pH 2.3 have been determined in three conditions: (i) 20 mM sodium phosphate buffer; (ii) 0.25 M NaCl; and (iii) 6.65 M GuHCl by small-angle X-ray scattering, and (iii) from translational diffusion coefficients measured by dynamic light scattering. The radius of gyration of the unfolded protein chain depends remarkably on the quality of the solvent, decreasing in the order 20 mM sodium phosphate greater than 6.65 M GuHCl greater than 0.25 M NaCl. The value of the radius of gyration in 0.25 M NaCl and also the value estimated for infinite ionic strength are close to the value predicted theoretically for the theta-point. This means that water in the absence of electrostatic interactions is a poor solvent for an unfolded protein while 6.65 M GuHCl is a better solvent.

Animals↗
PubMed1991

Measuring of the in-vitro assembly of microtubules by dynamic light scattering.

The kinetics of in-vitro assembly of microtubule protein (MTP) to microtubules (MTs) was followed under various conditions (temperature, GTP, ultrasonic treatment) by dynamic light scattering (DLS) and turbidimetric measurements. The results of both methods roughly coincide, but DLS additionally allows to differentiate between MTs and aggregates and to follow their growth. The complexity of these investigations, however, causes serious restrictions in the interpretation of the DLS data.

Animals↗
PubMed1990

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation.

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75-80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place. The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x 10(-7) cm2 . s-1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.

Circular Dichroism↗
PubMed1990

Insulin aggregation in solution.

The process of insulin aggregation in neutral solutions was studied by dynamic light scattering. Solutions of different concentrations were subjected to thermal and mechanical stress (37 degrees, rotation) for a period of 4 weeks. The starting solutions contained exclusively one particle distribution of insulin in the association equilibrium with hexamers as the largest structures. After a lag period of about 8 days the solutions showed continuously increasing scattering intensities but did not evolve perceptible turbidity within the experimental period. A more rapid increase in scattering intensity was observed in diluted than in concentrated solutions. The analysis of scattering data unexpectedly revealed that insulin species did not grow continuously. After the lag period one additional relatively restricted size distribution with particles of a mean radius of about 100 nm was found, the amount of which increased continuously with time. The occurrence of these particles seems to be related to adsorption phenomena of insulin to the solid interface. We assume the 100 nm-class of aggregates to be a transient state in the physical destabilization process of insulin solutions.

Humans↗
PubMed1988

Preparation and properties of large octylglucoside dialysis/adsorption liposomes.

The suitability and capacity of the polystyrene resin Wofatit EP 60 for the adsorption of octylglucoside, Triton X-100, Cholate, and CHAPS was studied. Optimal detergent/bead ratios and the maximum capacity of Wofatit EP 60 for the four detergents were determined as prerequisites for optimal application of the beads in liposome preparations. It is shown that large unilamellar liposomes can be prepared easily, quickly and cost-effectively using a combined dialysis/adsorption method with octylglucoside as detergent and Wofatit EP 60 and adsorbing polystyrene beads. Structure, composition, size, homogeneity, lamellarity, stability, internal volume, and residual octylglucoside concentration were studied by gel chromatography, radioactive assay, dynamic light scattering and electron microscopy. Vesicle size and homogeneity depend on lipid concentration, lipid composition, cholesterol content, and the rate of octylglucoside removal, but not on the detergent/lipid ratio. The reliability of the method and the properties of the vesicles are compared with those of other methods and researchers.

Adsorption↗
PubMed1986

Shape and quaternary structure of alpha-globulin from sesame (Sesamum indicum L.) seed as revealed by small angle x-ray scattering and quasi-elastic light scattering.

The alpha-globulin from sesame seed has a molar mass of 2.7 X 10(5) g mol-1, determined by x-ray scattering, and (2.8 +/- 0.3) 10(5) g mol-1, determined by quasi-elastic light scattering. The radius of gyration RG amounts to (4.1 +/- 0.1) nm and (3.9 +/- 0.2) nm as determined by Guinier approximation and from the distribution function D(x), respectively. The molecule has a Stokes radius Rs of (5.4 +/- 0.15) nm and a maximum dimension L of (11 less than L less than 15) nm. The translational diffusion coefficient D0(20),w and the ratio of fractional coefficients f/fmin amount to (3.95 +/- 0.12) X 10(-7) cm2 s-1 and 1.25, respectively. The quaternary structure of the protein molecule is approximated by a model consisting of six spherical subunits situated at the vertices of an octahedron having the symmetry 32.

Alpha-Globulins↗
PubMed1986

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering.

The structure of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500 +/- 4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume Vc, surface Oc, radius of gyration Rs, maximum dimension of the molecule L, thickness D, and cross section radius of gyration Rsq, agree within the experimental error limits. The mean values are Vc = 57 +/- 3 nm3, Oc = 165 +/- 10 nm2, Rs = 3.37 +/- 0.05 nm, L = 10.8 +/- 0.7 nm, D = 1.57 +/- 0.07 nm, Rsq = 0.92 +/- 0.01 nm. Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution functions. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D020, w = (5.9 +/- 0.2) X 10(-7) cm2 s-1 and (6.2 +/- 0.2) X 10(-7) cm2 s-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

A-form to A'-form conformational switch of double helices in rat liver 5S and 5.8S rRNA. Solution X-ray scattering evidence and circular dichroic measurements.

The wide-angle X-ray scattering of rat liver 5S rRNA and 5.8S rRNA molecules showed significant differences in the positions of the scattering maxima when dissolved in Mg2+-containing Tris/HCl buffer or in Mg2+-depleted buffer. A comparison of the experimental curves with theoretical curves calculated from atomic coordinates of double-helical models proved a switch from A form to A' form of the double-helical regions within the molecules by changing the buffer conditions. This result was supported by circular dichroic measurements. The A to A' transition may have important consequences for RNA-protein interactions.

Animals↗
PubMed1979

Spermine-DNA complexes build up metastable structures. Small-angle X-ray scattering and circular dichroism studies.

Spermine-DNA complexes have been examined by small-angle and wide-angle X-ray scattering as well as by circular dichroism studies. Condensed complexes are building up below a critical ionic strength. We have found that at one and the same ionic strength condensed complexes having two different supramolecular structures (Type I and Type II) can coexist. The structure of the condensates depends on the method of condensate formation. Phase transitions between these structures can be induced by thermal treatment. We conclude from these facts that with polyamine-DNA condensates metastable structures are of importance.

Animals↗
PubMed1978

Study of DNA-spermine interactions by use of small-angle and wide-angle X-ray scattering and circular dichroism.

Circular dichroism measurements with DNA-spermine complexes at 0.075 M NaCl and at 0.15 M NaCl reveal +psi (type I) and -psi (type II) CD spectra respectively. From small-angle X-ray scattering studies it could be shown that type I has a long-range order, short-range order supramolecular structure, while type II is of long-range disorder, short-range disorder structure. The secondary structure of the DNA in both types of condensates is B-like as concluded from wide-angle X-ray scattering diagrams of the condensates and from a comparison with the wide-angle X-ray curves of DNA and RNA in solution.

Animals↗
PubMed1978

[Secondary structure of condensed DNA. wide-angle, small-angle x-ray scattering and circular dichroism].

Ethanol precipitated DNA shows a CD spectrum of the +psi-type which is similar to that of DNA in the A-form. DNA condensed with cetyl-trimethylammonium-bromide shows, depending on the condensation velocity, a CD spectrum of the -psi-type, or a CD spectrum only slightly modified from that of DNA in solution. The first spectrum is similar to that of DNA in the C-form, and the second one, to that of DNA in the B-form. Using large-angle X-ray scattering of the three DNA condensates and comparing them with the scattering curves calculated from the atom coordinates for the A-, B-, and C-form of DNA it is shown that the secondary structure of the DNA belongs in all three cases to the B-family. It follows from this result that the secondary structure of DNA alone does not determine the type of CD spectrum. The CD spectrum of condensed DNA is essentially determined by the supramolecular structures of the partially crystalline DNA condensates. These supramolecular structures can be demonstrated by the small-angle X-ray diagrams. The condensation of DNA by ethanol and cetyl-trimethylammonium-bromide proceeds in the form of a partial crystallization of the DNA.

Animals↗
PubMed1977

[Diffuse x-ray wide-angle scattering of polyglutamic acid in solution].

The diffuse wide angle x-ray scattering (WAXS) of polyglutamic acid (PGA) in solution was studied using an x-ray diffractometer with small aperture of the primary beam. The scattering curve was recorded at an angular interval from (article: see text). The experimental scattering intensity of PGA with alpha-helical CD spectrum showed a maximum at 14.4 nm-1. Unordered PGA in solution yielded no maximum at this scattering angle. The studies have proved that the scattering theory can be applied to globular proteins in solution as well as to chain molecules in solution in this angular interval. The differences between the calculated scattering curves and the experimental curves indicate minor movements of the side chains of PGA in solutions and slight structuring of the solvent at the surface of the polypeptide chain.

Glutamates↗
PubMed1977

Substrate binding to solubilized cytochrome P-450 from rabbits at different temperatures.

The binding affinities of selected type I- and type II-substrates to partially purified cytochrome p-450 from rabbit liver microsomes were studied and found to differ from those of rats. The temperature dependence of the apparent binding constants qualitatively exhibited the same characteristics compared with that of rats. For type I-substrates endothermic and for type II-substrates exothermic reaction characteristics were observed. Taking into account the partition coefficients of the substrates so far investigated it is obvious that type I substrates with increasing hydrophobicity are bound more strongly while type II-substrates show a more complicated behvaiour. This may due to the fact that other types of binding are included besides the hydrophobic interactions.

Animals↗