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M H Koch

Publications and source records attributed to M H Koch.

At least 91 records · Page 5Linked to original sources
PubMed1990

An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC).

Structure and activation pattern of pyruvate decarboxylase (PDC) from yeast was studied by synchrotron radiation X-ray solution scattering. The results give a direct proof that the reversible deactivation of PDC at pH 8.0 is accompanied by the dissociation of the tetrameric holoenzyme into dimeric halves. The kinetics of this process was followed. At pH 6.5 the dimeric halves reassociate to a tetramer even in the absence of cofactors. The changes of the scattering pattern upon binding of the substrate-like activator pyruvamide indicate that the structure expands in the course of the enzyme activation.

Apoenzymes↗
PubMed1990

Changes in chromatin chain flexibility during condensation induced by sodium chloride, as evidenced by electric dichroism.

The electric linear dichroism of chicken erythrocyte chromatin has been measured as a function of NaCl concentration in the 1-28 mM ionic strength range, using a specially designed Kerr cell with reduced pathlength, and thus, smaller electrode surface. This allowed the determination of the dichroism of compact chromatin in conditions where artifacts due to possible contribution from turbidity are avoided, which was not the case for previous studies in the presence of di- or multivalent cations. The linear dichroism of compact chromatin was found to be positive, as expected from models of the 30-nm fibre in which the linker DNA runs perpendicular to the fibre axis. The dependence of the relaxation times on ionic strength reveals that the process of compaction is first accompanied by an increase in flexibility of the chain followed by a decrease, in the range of 5-10 mM NaCl, and a further decrease above 10 nM NaCl, corresponding to the compaction of the 30 nm fibre.

Animals↗
PubMed1990

Structure and assembly of calf hoof keratin filaments.

Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural parameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconstituted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solution scattering. The structures formed in solution at various stages of filament assembly were not sufficiently homogeneous to be studied by this technique. X-ray diffraction patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the patterns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof keratin filaments.

Animals↗
PubMed1989

Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small-angle X-ray diffraction.

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (greater than 90%) at different temperatures (20, 37, and 55 degrees C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20 degrees C, i.e. below the beta----alpha acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 degrees C), 4.60 nm for Re lipopolysaccharide (at 37 degrees C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37 degrees C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20 degrees C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1: square root of 2, 1: square root of 3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.

Escherichia coli↗
PubMed1989

Time-resolved synchrotron radiation X-ray solution scattering study of DNA melting.

Time resolved x-ray solution scattering measurements were made during thermal denaturation of DNA from various sources in the temperature range of 20-90 degrees C. Preliminary results on the influence of fragment length, ionic strength, and origin of the DNA on the time course of the scattering are described. Interpretation is based on model calculations of the scattering patterns. The results indicate that, for long DNA fragments at very low ionic strength, the melting process is a continuous phenomenon over the whole temperature range. It is accompanied by a progressive decrease of the radius of gyration of the cross section and of the mass per unit length. For short fragments of 146 base pair nucleosomal core DNA, stiffening of the DNA appears to precede a sharp melting transition.

Animals↗
PubMed1989

The superstructure of chromatin and its condensation mechanism. VI. Electric dichroism and model calculations.

Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations. Electric dichroism measurements on a complex of uncondensed chromatin with methylene blue were made to determine the contribution of the linker and of the nucleosomes to the total dichroism. A new approach to calculate the dichroism from realistic structural models, which also yields other structural parameters (radius of gyration, radius of gyration of the cross-section, mass per unit length) was used. Only a restricted range of structures is simultaneously compatible with all experimental results. Further, it is shown that previous interpretations of dichroism measurements on chromatin were in contradiction with X-ray scattering data and failed to take into account the distribution of orientation of the nucleosomes in the fibers. When this is done, it is found that the linker DNA in chicken erythrocyte and sea urchin chromatin must run nearly perpendicularly to the fibre axis. Taken together with the dependence of the fibre diameter on the linker length, these results provide the strongest evidence hitherto available for a model in which the linker crosses the central part of the fibre.

Animals↗
PubMed1989

Ehlers-Danlos syndrome type IV (EDS IV) as model of a defective biopolymer composite material.

The connective tissue of a lethal EDS IV case was investigated for the reasons of the manifested disturbances of the arterial wall. This functional disorder was attributed to the mechanical decoupling of elastin and collagen, with the premise of a composite material consisting of cellular, fibrillar, lamellar and other matrix components. A conceivable relation between the manifested deficiency of type III collagen and a disturbed anchoring of elastin is shown. These findings are supported by biochemical, morphological, x-ray and mechanical data.

Adult↗
PubMed1988

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates.

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.

Capsid Proteins↗
PubMed1988

Electric field x-ray scattering measurements on tobacco mosaic virus.

The feasibility of electric field x-ray solution scattering with biological macromolecules was investigated. Electric field pulses (1.25 to 5.5 kilovolts per centimeter) were used to orient tobacco mosaic virus in solution (4.5 milligrams per milliliter). The x-ray scattering is characteristic of isolated oriented particles. The molecular orientation and its field-free decay were monitored with a time resolution of 2 milliseconds by means of synchrotron radiation and a multiwire proportional area detector. The method should also be applicable to synthetic polymers and inorganic colloids.

Electricity↗
PubMed1988

The superstructure of chromatin and its condensation mechanism. V. Effect of linker length, condensation by multivalent cations, solubility and electric dichroism properties.

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb, Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg++ concentration at which precipitation occurs on chromatin concentration and confirm the differences between cations observed by X-ray scattering. The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.

Animals↗
PubMed1987

The superstructure of chromatin and its condensation mechanism. III: Effect of monovalent and divalent cations X-ray solution scattering and hydrodynamic studies.

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strengths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei. It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.

Animals↗
PubMed1987

The superstructure of chromatin and its condensation mechanism. IV. Enzymatic digestion, thermal denaturation, effect of netropsin and distamycin.

Changes in the structure of chicken erythrocyte chromatin fibres at low ionic strength resulting from enzymatic digestion, thermal denaturation and binding of Netropsin and Distamycin were monitored by synchrotron X-ray solution scattering. Digestion with micrococcal nuclease confirms the previous assignment of the 0.05 nm-1 band to an interference between nucleosomes with an average distance of 23 nm. The results of thermal denaturation indicate that above 40 degrees C there is a progressive increase of the internucleosomal distance and that above 60 degrees C the characteristic structure of the chromatin fibre is destroyed. Binding of Netropsin and Distamycin also results in an increase of the internucleosomal distance which can be estimated to correspond to about 0.2 nm/mol.

Animals↗
PubMed1987

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Synchrotron x-ray diffraction was used to identify the oligomers that formed during the earliest stages of actin polymerization. Solution diffraction patterns from G-actin (monomer) and from F-actin (polymer) contain information about the size and shape of actin monomers and the length, width, and subunit organization of filaments. Comparison of patterns collected during polymerization reveals an increase in scatter at spacings greater than 9.0 nm; formation of scattering bands at 5.4,4.9, and 3.4 nm; formation of a scattering minimum at 6.5 nm; and the presence of an isosbestic point at 9.0 nm. These scattering bands arise from the formation of, and organization of subunits in, filaments. At various actin concentrations (0.37-5 mg/ml), the change in scatter in these regions follows simple exponential kinetics with no detectable lag. Our analysis of the x-ray patterns shows that by 0.4 sec after mixing, most of the actin has formed dimers, which then rapidly incorporate into oligomers. At 4 mg/ml the early oligomers increase in length to greater than 30.0 nm within 10 sec. These results suggest that under our conditions actin molecules condense into filaments without the rate-limiting formation of nuclei.

Actins↗
PubMed1986

New aspects of the etiology of tendon rupture. An analysis of time-resolved dynamic-mechanical measurements using synchrotron radiation.

Native collagen fibers were exposed to different dynamic loads to simulate damage to tendons and ligaments relevant clinically and for sports medicine. The results suggest that the rupture of a tendon is caused at the submicroscopic fibrillar level. Not only slow or very fast elongation, but also very fast unloading of stretched fibers seems to be responsible for disseminated damage, which reduces the stability of a fiber. This damage is induced by intrafibrillar sliding processes, which occur only a few seconds before macroscopic slippage takes place. The significance of these events for the beginning and progress of repair in vivo is discussed. The conclusions are supported by simultaneous mechanical and radiological measurements, as well as by light- and electron-microscopic results.

Animals↗
PubMed1986

The superstructure of chromatin and its condensation mechanism. I. Synchrotron radiation X-ray scattering results.

Synchroton radiation X-ray scattering experiments have been performed on chicken erythrocyte chromatin fibres over a wide range of ionic conditions and on various states of the fibres (i.e. "native" in solution, in gels and in whole nuclei; chromatin depleted of the H1 (H5) histones and chromatin with bound ethidium bromide). A correlation between the results obtained with the various chromatin preparations provides evidence for a model according to which at low ionic strength the chromatin fibre already possesses a helical superstructure, with a diameter comparable to that of condensed chromatin, held together by the H1(H5) histone. The most significant structural modification undergone upon an increase of the ionic strength is a reduction of the helix pitch, this leads to condensation in a manner similar to the folding of an accordion. The details of this process depend on whether monovalent or divalent cations are used to raise the ionic strength, the latter producing a much higher degree of condensation. Measurements of the relative increase of the mass per unit length indicate that the most condensed state is a helical structure with a pitch around 3.0-4.0 nm. In this paper we give a detailed presentation of the experimental evidence obtained from static and time-resolved scattering experiments, which led to this model.

Animals↗
PubMed1986

The superstructure of chromatin and its condensation mechanism. II. Theoretical analysis of the X-ray scattering patterns and model calculations.

Model calculations on the superstructure of uncondensed and condensed chromatin are presented. It is found that agreement between the calculated X-ray solution scattering patterns and the experimental observations can be reached with the assumptions that: a) The uncondensed chromatin fibre in solution has a helix-like structure, with a pitch of ca. 33.0 nm, a helical diameter of ca. 20.0 nm and 2.75-3.25 nucleosomes per turn. b) The most condensed state of the chromatin fibre in solution is best represented by a helix-like structure with ca. 2.56 nucleosomes per turn, a pitch of ca. 3.0 nm and a helical diameter of ca. 27.0 nm.

Animals↗