PubMed Health⌕ Search

Biomedical subjects

R Misselwitz

Publications and source records attributed to R Misselwitz.

At least 19 recordsLinked to original sources
PubMed1992

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.

Amides↗
PubMed1992

Salt-dependent and protein-concentration-dependent changes in the solution structure of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis.

The solution structure of the histone-like DNA-binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg.ml-1, melting temperatures of 32 degrees C and 55 degrees C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic-strength-dependent differences in the temperature-induced unfolding of HBsu. Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2-3 mg.ml-1. Such protein-concentration-dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone-like DNA-binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst. Transition temperatures of 45 degrees C and 61 degrees C were found in differential scanning calorimetric measurements performed with HBsu in potassium-fluoride-free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.

Bacillus subtilis↗
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↗
PubMed1992

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.

Amino Acid Sequence↗
PubMed1992

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.

Amino Acids↗
PubMed1992

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.

Amino Acids↗
PubMed1990

Effect of platinum(II) chemotherapeutic agents on properties of DNA liquid crystals.

We have investigated the X-ray and optical properties (CD spectra and polarization microscopy) of liquid-crystalline phases and dispersions formed on pretreatment of low molecular weight DNA with the platinum(II) coordination complexes, cis-diammine-dichloroplatinum(II) (DDP), 2,2'-bipyridinedichloroplatinum(II) (1) and 2,2'-bipyridineethylenediammineplatinum(II) (2). It is demonstrated that the platination of DNA leads to the ordering of neighbouring molecules of DNA in liquid-crystalline phases being diminished. The intense bands observed in the CD spectra of liquid-crystalline dispersions prepared from DNA pretreated with 1 or 2 can be used to determine the orientation of the latter compounds with respect to the helical axis of the DNA and to detect distortions in the secondary structure of DNA. The possible causes of the appearance of the intense bands in the CD spectra of liquid-crystalline phases and alterations in the manner of packing of the molecules of DNA within them are discussed.

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↗
PubMed1979

The importance of the spin equilibrium in cytochrome P-450 for the reduction rate of the heme iron.

Based on the existence of a spin equilibrium of P-450 LM a thermodynamic model including the first two elementary steps has been developed. This model has been compared with a modified model of P-450 CAM first presented by SLIGAR [1]. For the quantitative description of the model of P-450 LM seven necessary constants have been calculated. In the model a 5-coordinated ferric heme complex as intermediate and precursor of the reduced state is included. The model is based on experimental data of the spin equilibrium and the binding of benzphetamine to P-450 LM2, on the redox potential of P-450 LM and further on the assumption of a structure of the precursor well adapted to reduction. The dissociation constant (KD) of the 6th heme iron ligand is calculated to be 0.03 meaning that only 3% of the high spin ferric state are 5-coordinated. From the thermodynamic parameters of the model the sequential order of the reaction process can be derived and conclusions can be drawn on possibly reaction controlling steps. Experimentally it is shown that the substrate induced spin shift is correlated with the reduction rate. This has been shown to be valid in dependence on the degree of saturation with only one substrate as well as with different substrates indicating that independent of the individual substrate only the shift of the spin state controls the reduction. From the experiments as well as from the model it can be concluded that P-450 works in the first reaction step in two states (different in structural and functional properties) being in equilibrium with each other. Different affinities of these two states towards the substrate shift the equilibrium. This shift increases the concentration of the reducible intermediate and by this the reduction as second elementary step is regulated.

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

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↗
PubMed1976

Circular dichroism studies on DNA condensed in NaCl cetyltrimethyl ammonium bromide solutions.

It is shown by means of circular dichroism studies of variously condensed forms of DNA that the specific supramolecular structure of DNA determines the type of CD spectra. DNA, condensed (crystallized) slowly in the presence of cetyltrimethyl ammonium bromide yields a spectrum very similar to that of DNA in solution in the B-form. The condensates appear in the phase-contrast microscope as spherulitic crystallites. Rapidly condensed DNA in the presence of cetyltrimethyl ammonium bromide shows a spectrum of the psi-type with large negative ellipticites. The influence of condensation velocity upon the supramolecular structure of DNA gives evidence that the various condensation forms of DNA are not thermodynamical equilibrium conformations.

Bromides↗
PubMed1976

Folding crystallization of DNA. Circular dichroism studies.

CD spectra of DNA monocrystals are extremely different from spectra of psi-DNA or DNA-histone H1 or DNA-polylysine complexes. They are discussed to be dependent on the supramolecular organization of DNA in the condensed form, and they are not in contradiction to the previously proposed seven folding model of DNA in chromatin. Conclusions are drawn with regard to the interpretation of chromatin CD spectra.

Chromatin↗
PubMed1976

Differentiation between type I and type II substrate binding to cytochrome P-450 by variation of temperature.

The binding affinities of type I- and type II-substrates to cytochrome P-450 solubilized from phenobarbital induced rat liver microsomes in dependence on the temperature have been determined. Both classes of substrates have been found to exhibit different temperature behaviour. The tendency of type I-substrates (benzphetamine and hexobarbital) to form complexes increases with increasing temperature; type II-substrates show the inverse tendency. From the van't Hoff plot the binding enthalpy was calculated and discussed in connection with the entropy and free enthalpy values. These data ascertain the suggestion of different binding sites for both classes of substrates.

Aniline Compounds↗