Helmut Durchschlag

Universität Regensburg | UR · Institute of Biophysics and physical Biochemistry

Modeling simple and complex biopolymers in solution requires the shapes of these molecules to be approximated by bead modeling procedures, primarily for the prediction of hydrodynamic and scattering quantities. Though several bead modeling strategies (strict, shell and filling models) and a variety of computer programs (preferably the HYDRO suite b...

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of sali...

The evolution of the prototypical (beta alpha)(8)-barrel protein imidazole glycerol phosphate synthase (HisF) was studied by complementary computational and experimental approaches. The 4-fold symmetry of HisF suggested that its constituting (beta alpha)(2) quarter-barrels have a common evolutionary origin. This conclusion was supported by the comp...

The shape of macromolecules can be approximated by filling models, if both hydrodynamic and scattering properties should be predicted. Modeling of complex biological macromolecules, such as oligomeric proteins, or of molecule details calls for usage of many beads to preserve the original features. However, the calculation of precise values for stru...

Hydrodynamic models of proteins have been generated by recourse to crystallographic data and applying a filling model strategy in order to predict both hydrodynamic and scattering parameters. The design of accurate protein models retaining the majority of the molecule peculiarities requires usage of many beads and consideration of many serious prob...

The shape of simple and complex biological macromolecules can be approximated by bead modeling procedures. Such approaches are required, for example, for the analysis of the scattering and hydrodynamic behavior of the models under analysis and the prediction of their molecular properties. Using the atomic coordinates of proteins for modeling inevit...

The present knowledge of protein science includes information on amino acid sequence and 3D structure in terms of precise models on the atomic level. Recourse to the respective databanks and advanced computer programs allows aseries of molecular features to be calculated. Application of analytical surface calculation programs (SIMS, MSRoll) based o...

Crystallographic or NMR techniques provide information on the precise 3D structure of proteins and reveal the position of some water molecules preferentially bound to certain amino acids. Knowledge of the hydration sites is required for understanding behavior and interactions of hydrated proteins in context with flexibility, dynamics and functional...

Self-assembling systems composed of organic molecules and metal ions represent one of the topics in modern supramolecular chemistry. Up to now, their physical characterization was essentially limited to crystals or to surface layers. Studies on solubilized systems, e.g., on their molecular mass distribution or association behavior, are rare. We hav...

Conformational changes of citrate synthase upon ligand binding and upon denaturation were monitored in solution by spectroscopic techniques, analytical ultracentrifugation and small angle x-ray scattering. Mafor contributions to structural alternations and to the stabilization against denaturation are attributable to the substrate oxaloacetate

A calculation procedure is presented which relates solution scattering and hydrodynamic parameters. Biopolymers are modeled by whole-body approaches, approximating their overall shape by spheres or prolate/oblate ellipsoids of revolution. Molar masses, partial specific volumes, radii of gyration, volumes and surface-to-volume ratios are used for pr...

Knowledge of the structure of protein-SDS complexes and the constituent free proteins and SDS micelles is important for our understanding of protein-detergent interactions and for the application and improvement of physicochemical techniques connected with the purification and characterization of proteins. Several reasons are responsible for presen...

Crystallins, the major eye lens proteins, were investigated after preceding irradiation with X-rays or UV light by using analytical ultracentrifugation and other physicochemical and analytical techniques. Sedimentation velocity and sedimentation equilibrium runs of unirradiated and irradiated samples of the crude extract and individual fractions of...

Several enzymes have been irradiated with x-rays and UV-light in aqueous solution. Various aspects of radiation damage have been studied systematically by a series of solution techniques. The radiation action may be modified effectively by many protectants.

Bead models, composed of overlapping spheres of different or identical radii, were generated from the crystal structures of selected proteins (citrate synthase, lactate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase). Starting from the atomic coordinates taken from the Brookhaven Protein Data Bank, different methods (“running mean” or...

Small-angle X-ray scattering was applied to study the structure of horse-spleen ferritin and apoferritin as well as of various intermediate states differing in their iron content. The intermediates were produced by partial removal of iron from ferritin or by incorporation of iron into apoferritin. Measurements were performed in dilute buffer and in...

Binding of variable amounts of sodium dodecyl sulfate (SDS) to bovine serum albumin (BSA) may be followed by analytical ultracentrifugation. The molar masses of the protein-surfactant complexes obtained from sedimentation equilibria allow binding isotherms to be established. UV absorption spectroscopy, fluorescence and circular dichroism are used f...

A novel, universal and easy-to-use approach is presented which allows the ab initio calculation of partial volumes of organic compounds, valid for aqueous solutions at 25°C. The method is based on Traube’s additivity principle and concept of volume increments for atoms. His concept, however, was improved considerably by manifold adaptations, correc...

The technique of small-angle scattering can be used advantageously for deriving the shape of proteins in solution. Presently, advanced ab initio approaches allow the reliable automated reconstruction of particle shapes. In particular, the procedures based on simulated annealing such as DAMMIN in combination with tight constraints turn out to be hig...

The X-ray damage of the sulfhydryl enzyme malate synthase from yeast has been investigated by physicochemical studies including time-resolved small-angle X-ray scattering. Our previous studies already suggested the role of cysteine-sulfenic acid and disulfides in the damage process and established a simple model for the occurring aggregation. The p...

The reconstruction of sphere shells as realized in bacteriophage capsids can be achieved by ab initio modelling approaches based on a genetic algorithm or simulated annealing. The application of tight constraints such as icosahedral symmetry makes the DAMMIN procedure the method of choice. The ab initio models obtained may be compared with three-di...

Various fractions of poly-m-bromostyrene and poly-o-bromostyrene in benzene were studied by the small-angle and wide-angle x-ray methods. The combination of the two methods makes it possible to supply information on short-range order and long-range order in the same molecule. Four fractions of poly-m-bromostyrene of the approximate ratio of the deg...

Spectroscopic techniques (UV absorption, fluorescence and circular dichroism) are applied for probing the conformational stability of lysozyme as a model protein after the impact of surfactants. The investigations allow the equilibrium constant, K, and the free energy change, ΔG, of the transition from the folded (native) to the unfolded (denatured...

The joint use of small-angle X-ray scattering (SAXS) and hydrodynamic data permits biologically useful reconstructions of protein structures to be determined. Low-resolution shapes of proteins can be obtained by SAXS-based modeling approaches, among them the ab initio approaches being the most recent and challenging ones. The programs DAMMIN and GA...

For modeling the low-resolution shape of the dodecameric subunit of Lumbricus terrestris hemoglobin, experimental small-angle X-ray scattering (SAXS) data and ab initio modeling approaches using agenetic algorithm or simulated annealing have been applied. In addition to the use of strict ab initio approaches, procedures which additionally include a...

Three-dimensional low-resolution models of the biochemically and biotechno- logically significant enzyme cellobiose dehydrogenase (CDH) were predicted by advanced modeling approaches based on experimental one-dimensional low-resolution small-angle x-ray scattering (SAXS) data. In particular, the ab initio modeling program DAMMIN by Svergun was used...

この論文は国立情報学研究所の電子図書館事業により電子化されました。 研究会報告 Water is recognized to have many important roles in influencing protein structure, folding and function. Understanding protein hydration requires the elucidation of the effects of both the solvent and the protein. While a variety of physical techniques (e.g., X-ray crystallography, small-angle X-ray scatterin...

The combination of high-speed sedimentation equilibria and specific volume estimates under isomolal or isopotential conditions allows the molar mass of proteins, denatured by chaotropic reagents such as guanidinium chloride (GdmCl) or ionic detergents such as sodium dodecyl sulfate (SDS), to be analyzed. The results depend strictly on the procedure...

The information provided by small-angle X-ray scattering, electron microscopy and hydrodynamics for the giant hemoglobins from different annelid species (Lumbricus terrestris, Macrobdella decora,Eudistylia vancouverii) has been compared. The results reveal that these hemoglobins exhibit slight differences in their fine structure, particularly with...

The atomic coordinates of proteins, for example, of the enzyme lysozyme as a small model protein, can be used for calculating analytically the exact protein surface in terms of dot surface points and for visualizing both the atoms and the surface points. On the basis of known hydration numbers for the constituent amino acid (AA) residues as suggest...

Annelids possess giant extracellular oxygen carriers that exhibit a hexagonal bilayer appearance and have molecular masses of approximately 3.5 MDa. By small angle x-ray scattering (SAXS), Eudistylia vancouverii chlorocruorin and Macrobdella decora hemoglobin were investigated in solution. On the basis of the experimental SAXS data, three-dimension...

The implications of protein-water interactions are of importance for understanding the solution behavior of proteins and for analyzing the fine structure of proteins in aqueous solution. Starting from the atomic coordinates, by bead modeling the scattering and hydrodynamic properties of proteins can be predicted reliably (Debye modeling, program HY...

The radiation damage of lysozyme has been investigated in aqueous solution, after preceding X- and UV-irradiation in the absence or presence of various additives. Activity measurements and UV absorption and fluorescence spectroscopy turned out to be powerful screening techniques for disclosing the damages (inactivation, aggregation, loss of aromati...

Reconstructions of low-resolution three-dimensional solution structures of proteins from one-dimensional small-angle X-ray scattering (SAXS) data can be achieved by trial-and-error or ab initio approaches. It is demonstrated that all tested advanced approaches (GA, DAMMIN, SAXS3D) reproduce the shape of three selected test proteins (malate synthase...

Small-angle x-ray investigations on poly-o-bromostyrene in benzene solution at extremely high resolution (corresponding to a Bragg's value of 3200 Å) proved to be able to link together the ranges covered by small-angle x-ray scattering and light scattering. On the other hand, there are problems encountered in molecular weight determination of chain...

Summary This document is part of Subvolume A ‘Structural and Physical Data I’ of Volume 2 ‘Proteins - Biochemical and Physical Properties’ of Landolt-Börnstein - Group VII Biophysics.

Summary This document is part of Subvolume A ‘Structural and Physical Data I’ of Volume 2 ‘Proteins - Biochemical and Physical Properties’ of Landolt-Börnstein - Group VII Biophysics.

Summary This document is part of Subvolume A ‘Structural and Physical Data I’ of Volume 2 ‘Proteins - Biochemical and Physical Properties’ of Landolt-Börnstein - Group VII Biophysics.

Summary This document is part of Subvolume A ‘Structural and Physical Data I’ of Volume 2 ‘Proteins - Biochemical and Physical Properties’ of Landolt-Börnstein - Group VII Biophysics.

Summary This document is part of Subvolume A ‘Structural and Physical Data I’ of Volume 2 ‘Proteins - Biochemical and Physical Properties’ of Landolt-Börnstein - Group VII Biophysics.

The solution structure of simple and complex proteins can be modeled efficaciously, starting either from solution scattering data or from the 3D structures derived from crystallographic work or from electron microscopy. Best fitting low-resolution bead models of proteins in solution can be retrieved successfully from the experimental scattering pro...

Suitable supramolecular building blocks can assemble via non-covalent interactions into large, often regular structures, such as surface films. Uniformity of the state of association of the building blocks seems to be a prerequisite for the success of the assembly process. The paper shows how the association behavior of the compounds can be charact...

Starting from the atomic coordinates of proteins, by modern surface calculation programs the exact surface topography of proteins can be visualized in terms of dot surface points. In combination with tricky hydration algorithms, based on the hydration numbers of individual amino acid (AA) residues and appropriate selection criteria, this may be use...

The solution behavior of proteins is governed by their molecular characteristics and the water molecules that bind to the protein surface. Based on the D structure of proteins, scattering and hydrodynamic modeling approaches may be applied to simulate both the protein and the water moieties realistically. Computer simulations make use of different...

Whole-body and multi-body approaches as well as empirical relations allow the establishment of manifold correlations between structural and hydrodynamic parameters of biopolymers. Hydrodynamic properties can be predicted reliably from crystallography, small-angle X-ray scattering or other structural data. The inverse procedure, the prediction of st...

The 3.5-MDa extracellular hemoglobin from Lumbricus terrestris is a giant heteromultimeric hexagonal bilayer complex. The consensus model of the complex as derived from small-angle X-ray scattering (SAXS) and a 3D reconstruction obtained from cryoelectron microscopy (EM) were used as bases for the prediction of hydrodynamic parameters (sedimentatio...

The specific volume of charged supramolecular compounds dissolved in organic solvents varies considerably with the solvent system applied; in addition, it is influenced by the presence of salt. In this study we determined the specific volume of an uncharged molecule from the same molar mass range in order to find out whether it shows the same depen...

Hydration contributions of proteins can be taken into account by advanced modeling techniques starting from the atomic-level structure. Bead modeling may be used both for individual amino acid residues and individual water molecules placed at preferred positions on the protein surface. The exact calculation of the molecular volumes and surfaces of...

The comparative investigation of biopolymer hydration by physicochemical techniques, particularly by small-angle X-ray scattering, has shown that the values obtained differ over a wide range, depending on the nature of the polymer and the environmental conditions. In the case of simple proteins, a large number of available data allow the derivation...

The investigation of detergents and protein–detergent complexes by UV–Vis absorption spectroscopy, fluorescence excitation and emission and near- and far-UV circular dichroism yields a variety of molecular characteristics, provided the conventional techniques are modified in an appropriate manner. In the case of weakly absorbing detergents, labelin...

The molecular properties of proteins and other biomolecules have been investigated in aqueous solution, after preceding X- or UV-irradiation. Spectroscopic techniques (UV–Vis absorption, fluorescence, circular dichroism), size-exclusion chromatography, and other physicochemical techniques were applied to differentiate between various radiation effe...

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunit of molecular mass 32 kDa. The enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. Oligomerization is required for enzyme activity and thermostability. We report here on a subunit interface mutat...

Hydrodynamic multibody models (bead models) of selected proteins were derived from crystal, SAXS and electron microscopic structures, by using various data reduction procedures. Pair distance distribution functions and scattering curves of initial and reduced models were calculated in order to check the maintainance of the structural characteristic...

Investigations of monomeric and micellar detergents, protein–detergent complexes, as well as native and denatured proteins by means of various physicochemical techniques yield a wide range of molecular characteristics of the components under analysis. Varying the experimental conditions (e.g., the concentration of solutes or the ionic strength of t...

Analytical ultracentrifugation was used to study two different types of supramolecular assemblies, namely, a cobalt coordination array based on metal-ligand interactions and a calixarene capable of self-association via hydrogen bonds. The state of association of the two supramolecular compounds studied was shown to be fully and reliably characteriz...

Selected nonionic and zwitterionic detergents have been investigated by sedimentation velocity and equilibrium runs in the analytical ultracentrifuge in a systematic manner. UV-visible absorption spectroscopy and determinations of the partial specific volumes were used as complementary techniques. Pilot tests comprised the following detergents: Non...

The performance of different expressions for the hydrodynamic interaction tensor in the prediction of translational and rotational friction coefficients and intrinsic viscosities of a broad spectrum of different models consisting of two unequal spheres, varying from separated to completely overlapping, was tested in a systematic manner. The emphasi...

The most important water-soluble components of the vertebrate eye (lens proteins, aqueous humor, vitreous, hyaluronic acid, ascorbic acid) have been investigated in aqueous solution, after preceding X- or UV-irradiation. Spectroscopic, chromatographic, electrophoretic, hydrodynamic and analytic techniques have been applied, to monitor several radia...

For correlating a number of structural and hydrodynamic parameters, biopolymers were modeled as prolate or oblate ellipsoids of revolution or as spheres (whole-body approaches). Axial ratios were estimated by combining the radius of gyration with either the hydrated volume or the surface-to-volume ratio. From these axial ratios and other parameters...

Volumetric properties of macromolecules and low-molecular compounds are necessary auxiliary means for the determination of molar masses from solution-scattering and hydrodynamic techniques. In many cases experimental determinations of partial volumes can or have to be replaced by calculative procedures. A universal approach for the calculation of b...

The most popular method for estimating the molecular weight of a polypeptide chain is the measurement of its mobility by polyacrylamide gel electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate. The application of the method to glycoproteins or other conjugated proteins, however, poses several problems, primarily due to th...

Several approaches have been applied to establish correlations between crystal structure analysis, small-angle scattering, sedimentation/diffusion, and viscosity data. Agreement has been reached between the various solution data, and also with results from crystallography if hydrational contributions are considered. As illustrative examples, the st...

The radiation damage of several proteins has been investigated in aqueou solution, after preceding X- or UV-irradiation. In order to differentiate between different radiation effects, a series of physico-chemical and biochemical methods as well as novel evaluation and normalization procedures have been applied. Numerous changes of several molecular...

Analytical ultracentrifugation is a powerful tool for investigating the size of proteins in solution, especially by measuring sedimentation and diffusion coefficients and molar masses. Several further molecular parameters such as frictional ratios, axial ratios of hydrodynamic models, and Stokes radii allow a rough estimate of the protein overall s...

In the past the technique of small-angle scattering has been a powerful tool for studying conformational changes of proteins which occur, for example, upon binding with ligands. Results obtained by different authors from X-ray and neutron experiments on a variety of proteins and under various conditions have been compiled. This offers the possibili...

The particle weight of 21 glycoproteins has been determined by SDS-PAGE and high-speed sedimentation equilibrium, focussing on the influence of carbohydrate content, partial specific volume ( [`(v)]2\bar v_2 ), and relative molar mass (M r) on the results of both techniques. Results were compared with literature data and with 22 nonconjugated prote...

The investigation of sulfur-containing biomolecules, especially of sulfhydryl proteins, is of particular interest in radiation biology. Sulfhydryl enzymes are useful objects for studying both structural and functional changes caused by radiation. In this context oxidation of enzyme sulfhydryls, inactivation (continuing in the post-irradiation phase...

The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues). Radiation-induced changes of enzymic activity were registered immedi...

Specific volume quantities of macromolecules are of importance in connection with a variety of physico-chemical and biochemical techniques, such as analytical ultracentrifugation and small-angle scattering, e.g., for the determination of molecular weights in two- and multicomponent solutions. Specific volumes may be obtained from density experiment...

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsib...

The X-ray induced aggregation of the sulfhydryl enzyme malate synthase in aqueous solution was monitoredin situ by time-resolved small-angle X-ray scattering. Experiments were performed in the absence/presence of various additives: formate, superoxide dismutase, catalase, NaCl, acetyl-CoA, glyoxylate, malate, pyruvate, O2[`.]O_2^{\bar .} and H2O2),...

The partial specific volume, \(\bar v_2 \), represents an essential property of macromolecules which is of particular significance for applying a variety of physicochemical and biochemical techniques. The most prominent example is the determination of molecular weights of macromolecules in simple two-component solutions from ultracentrifugal data,...

The sulfhydryl enzyme malate synthase from baker’s yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: ≃ 10 mg/ml; volume: 120 μl), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for...

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role...

The sulfhydryl enzyme malate synthase was inactivated in air-saturated aqueous solution by X-irradiation (2 kGy). Changes of activity were registered up to 60 h after irradiation. Effects of specific additives (formate, superoxide dismutase, catalase), added before and/or after irradiation, revealed the role of the deleterious radical and non-radic...

The apparent partial specific volume φ of proteins in multicomponent solutions may exhibit marked differences from those of proteins in simple two-component systems. Therefore, explicit knowledge of φ is of great importance in connection with a wide variety of biochemical techniques, especially those which involve conditions causing protein denatur...

The partial specific volume of proteins has been investigated as a function of protein concentration, temperature, pH, and a number of denaturing and non-denaturing solvent components. Protein concentration (5–100 mg·ml−1) is found to have no effect; increasing temperature (4–40°C), as well as addition of electrolytes, sugars, and polyols, leads to...

Without Abstract

The sulfhydryl enzyme malate synthase was shown to undergo an X-ray induced aggregation and inactivation in solution (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). Further evidence for the occurrence of aggregation and inactivation and also of fragmen­ tation and partial unfolding of the enzyme upon X-irradiation was obtaine...

The X-ray induced inactivation and aggregation of the enzyme malate synthase in aqueous solution were investigated by small-angle X-ray scattering and enzymic tests. Enzymic activity decreases about exponentially with increasing dose. The rate of inactivation depends linearly on the dose rate; the extrapolation to zero dose rate yielded a finite li...

1. Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity. About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts. The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. 2. Sedimentation a...

Malate synthase undergoes an X-ray induced aggregation which can be monitored in situ by small-angle X-ray scattering; the analysis of scattering curves, taken at subsequent stages of aggregation, has led to the establishment of a tentative model for an aggregation in two dimensions (Zipper and Durchschlag (1980) Rad. and Environm. Biophys., in pre...

Small-angle X-ray scattering measurements on malate synthase in aqueous solution revealed a continuous increase of the intensity in the innermost portion of the scattering curve with increasing measuring time. We have definite evidence that this increase reflects an X-ray induced aggregation of the enzyme particles in the course of the small-angle...

The X-ray induced aggregation of ribonuclease, lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and serum albumin in aqueous solution was monitored in situ by means of small-angle X-ray scattering. The time dependence of several molecular parameters viz. the apparent radius of gyration\(\tilde R\), the maximum visible d...

Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles durin...