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H Welfle

Publications and source records attributed to H Welfle.

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

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

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↗
PubMed1989

Infrared data indicate U.U mispairs in rat liver 5S RNA.

The infrared spectra of ribosomal 5S RNAs of Escherichia coli and rat liver were measured under conditions where the structure of 5S RNA is preserved. The comparison of the spectra shows significant spectral differences for the U residues which resemble in their characteristics the infrared spectra of polyuridylic acid in its base paired state. This strongly indicates the presence of U.U mispairs in rat liver 5S RNA. Juxtaposed U residues at positions U80-U96 and U73-U102 are potential candidates for U.U mispairs in rat liver 5S RNA.

Animals↗
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

Protein--RNA interaction in the rat liver 5S rRNA-protein L5 complex studied by digestion with ribonucleases.

Protein-RNA interactions in the 5S rRNA-protein L5 complex from rat liver ribosomes were studied by limited digestion of free and protein bound 5S rRNA with ribonuclease A and T1. In the complex with protein L5 the digestion of 5S rRNA by ribonuclease T1 is decreased at G37 and G89, whereas U38 and C39, and to a lower extent also C10 and U12 become accessible for ribonuclease A.

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↗
PubMed1985

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA.

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

Adenine↗
PubMed1978

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions.

1. By enlarging the dimensions of the gels used in the usual analytical two-dimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two-dimensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis, respectively. 24 proteins of the small and 24 proteins of the large ribosomal subunit were isolated in pure form. 4. The amino acid compositions of 24 proteins of the small and of 19 proteins of the large subunit were determined.

Amino Acids↗
PubMed1978

Number and molecular weights of the basic proteins of rat liver ribosomes.

1) Efficient separation of the proteins from rat liver ribosomes can by achieved by two-dimensional polyacrylamide gel electrophoresis. Complete separation of all components, however, is not possible with one system only. Comparison of the results obtained with different systems suggests further heterogeneity of S15, L22, L28, L33 and L35 and enables identification of S15a, S15b, L22a, L22b, L28a, L28b, L33, L33a, L35a and L36b. 2) Ribosomal proteins were substituted with iodoacetamide prior to electrophoresis or handled in all steps of the procedure in the presence of reducing agents. These procedures prevent the formation of oxidation products described erroneously as ribosomal proteins S5, S6, L15, L17 and L32 in earlier papers. 3) Estimation of the molecular weights was performed by two-dimensional separation of the small and large subunit proteins using sodium dodecyl sulphate in the second dimension. The positions of the 70 basic proteins in the 2-D patterns were identified. 4) The small and large subunit proteins have molecular weights in the range of 8000 to 35,000 and 11,000 to 55,500 Dalton, respectively. The number average molecular weights for the small and large subunit proteins are 22,500 and 26,500 Dalton, respectively. The sum of the molecular weights is 0.67 x 10(6) Dalton for the proteins of the small subunit and 1.05 x 10(6) Dalton for the proteins of the large subunit.

Acetates↗
PubMed1977

Presence of an endopeptidase activity in rat liver ribosomes.

Preparation of ribosomes using different procedures (treatment of postmitochondrial-postlysosomal supernatant or microsomes with 1% triton in 0.15 or 0.5 M KCl and subsequent sucrose gradient centrifugation; treatment of microsomes with 1.5% deoxycholate/2% triton) results in purified ribosomes which contain an endopeptidase activity detectable by breakdown of ribosomal proteins to trichloroacetic acid soluble split products. The proteolytic activity can be recovered also in the extracted proteins of whole ribosomes. With ribosomes the pH optimum of proteolytic breakdown is at about 7. The inhibition of the activity by leupeptin, DIFP and soya bean trypsin inhibitor suggests a serine type of the proteolytic activity.

Animals↗
PubMed1976

Ionic interactions in eukaryotic ribosomes: splitting of the subunits of rat liver ribosomes by treatment with monovalent cations.

Proteins of rat liver ribosomal subunits were successively split off by stepwise treatment of the particles with various concentrations of LiC1 (0.4--1.0 M) or KC1 (0.6--1.5 M). Split proteins and core particle proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The LiC1 concentrations were estimated at which the individual proteins are split off. The proteins of 40S and 60S subunits were classified in 4 and 5 groups, respectively, according to their splitting behavior. The effect of LiC1 and KC1 is similar, but higher concentrations of KC1 than of LiC1 are required to obtain comparable split protein patterns. Evidence for structural changes of the core particles during the split procedures was obtained.

Animals↗