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M H Koch

Publications and source records attributed to M H Koch.

At least 55 records · Page 3Linked to original sources
PubMed1998

A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis.

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1998

Complex melting of semi-crystalline chicory (Cichorium intybus L.) root inulin.

When concentrated solutions (30-45% by weight) of inulin (degree of polymerization 2-66, number average degree of polymerization 12) are cooled at 1 degree C/min or 0.25 degree C/min from 96 degrees C to 20 degrees C, suspensions of semi-crystalline material in water are formed. A thermal nucleation process was detected by optical microscopy: the 8-like shaped crystallites resulting from primary nucleation at higher temperature are larger than those resulting from secondary nucleation at lower temperature. Differential scanning calorimetry (DSC) thermograms display melting profiles with three to four partly overlapping endotherms that vary as a function of concentration, cooling rate during crystallization and storage time at 25 degrees C of the crystallite suspension. Recrystallization during melting was observed. The wide-angle X-ray scattering patterns of the samples at 25 degrees C correspond to those of the hydrated crystal polymorph. The structural changes during melting indicated the existence of a single crystal polymorph throughout melting. A periodicity of 95 A, arising from alternating regions of different electron density, is detected in the small angle X-ray scattering patterns at 25 degrees C. The stepwise increase of the long period upon heating is related to the existence of two types of lamellar stacks: one with a higher long period, resulting from the primary nucleation and thus crystallized at high temperature, and a second one with a smaller long period, formed by crystallization at lower temperature. The lamellae formed at low temperature melt at a lower temperature than those formed at high temperature, explaining the existence of the two DSC-endotherms.

Calorimetry, Differential Scanning↗
PubMed1998

Characterization of the nonlamellar cubic and HII structures of lipid A from Salmonella enterica serovar Minnesota by X-ray diffraction and freeze-fracture electron microscopy.

The aggregate structures of lipid A, the 'endotoxic principle' of bacterial lipopolysaccharide (LPS), from rough mutant Salmonella enterica sv. Minnesota R595 was analyzed at different water content, cation (Mg2+) concentration, and temperature applying synchrotron radiation X-ray diffraction and, in selected cases, freeze-fracture electron microscopy. The X-ray diffraction spectra prove the existence of different lamellar, mixed lamellar/cubic, various cubic, and inverted hexagonal (HII) structures depending on ambient conditions. The three mainly bicontinuous cubic phases Q224, Q229, and Q230 can be observed between 30 and 50 degrees C in narrow water and cation concentration ranges. Above 50 degrees C, Q212 an intermediate phase between bicontinuous and micellar is adopted. In freeze-fracture electron microscopic experiments, cubic structures of these symmetries are not readily detected, which can be understood in the light of changes in hydration during freezing and the metastability of these phases. However, 'lipidic particles' closely related to cubic phases are observed. Above 65-70 degrees C, the existence of the HII phase with hexagonal periodicities dH between 4.0 and 6.0 nm for different hydration states is shown using both techniques. Possible biological implications for the preference of lipid A for nonlamellar structures are discussed.

Endotoxins↗
PubMed1997

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models.

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

Deuterium↗
PubMed1997

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. II. A model of the ribosome and its RNA at 3.5 nm resolution.

Selectively deuterated 70 S E. coli ribosomes and isolated 30 S and 50 S subunits were analyzed by X-ray and neutron solution scattering. The resulting contrast variation data set (42 curves in total) was proven to be consistent in describing the ribosome as a four-phase system composed of the protein and rRNA moieties of both subunits. This data set thus provides ten times more information than a single scattering curve. A solid body four-phase model of the 70 S ribosome at low resolution was built from the envelope functions of the 30 S and 50 S subunits and of those of the corresponding RNA moieties. The four envelopes were parameterized at a resolution of 3.5 nm using spherical harmonics and taking into account interface layers between the phases. The initial approximation for the envelopes of the subunits was taken from electron microscopic data presented recently by J. Frank and co-workers (Albany); the rRNA envelopes were initially approximated by spheres. The optimization and the refinement of the model proceeded by non-linear least squares minimization fitting the available experimental data. The refined envelopes of the subunits differ by about 10% from the starting approximation and the shape of the final 70 S model lies between the outer envelopes of the models by Frank and by M. von Heel & R. Brimacombe (Berlin). The rRNA moiety in the 30 S subunit is more anisometric than the subunit itself, whereas the rRNA of the 50 S subunit forms a compact core. The rRNAs protrude to the surfaces of the subunits and occupy approximately 30 to 40% of the corresponding surface areas. X-ray scattering curves of the two main functional elongation 70 S complexes (pre- and post-translocational) differ only marginally from those of the non-programmed ribosomes, suggesting that the low resolution four-phase model is also valid for the elongating 70 S ribosome.

Escherichia coli↗
PubMed1997

The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy.

The tertiary structural changes occurring during the photocycle of bacteriorhodopsin (BR) are assigned by X-ray diffraction to distinct M states, M1 and M2. Purple membranes (PM) of the mutant Asp96Asn at 15, 57, 75 and 100% relative humidity (r.h.) were studied in a parallel X-ray diffraction and Fourier transform infrared (FTIR) spectroscopic investigation. Light-dependent conformational changes of BR-Asp96Asn are observed at high hydration levels (100 and 75% r.h.) but not in partially dehydrated samples (57 and 15% r.h.). The FTIR spectra of continuously illuminated samples at low and high hydration, despite some differences, are characteristic of the M intermediate. The changes in diffraction patterns of samples in the M2 state are of the same magnitude as those of wild-type samples trapped with GuaHCl in the M(G) state. Additional large changes in the amide bands of the FTIR spectra occur between M2 and M(G). This suggests, that the tertiary structural changes between M1 and M2 are responsible for the switch opening the cytoplasmic half-channel of BR for reprotonation to complete the catalytic cycle. These tertiary structural changes seem to be triggered by a charge redistribution which might be a common feature of retinal proteins also in signal transduction.

Amino Acid Sequence↗
PubMed1997

Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state.

Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T-->R transition observed in the crystals. Taking the crystal R structure as a-reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11 degrees in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9 degrees around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis.

Allosteric Site↗
PubMed1996

Structural model of the 50S subunit of E. coli ribosomes from solution scattering.

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Based on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40 A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Escherichia coli↗
PubMed1996

Small-angle X-ray scattering of kappa- and iota-carrageenan in aqueous and in salt solutions.

Aqueous and saline (0.03-0.08 M NaCl) solutions of the Na(+)-salt of kappa- and iota-carrageenan (3-17 mg/ml) have been investigated using small-angle X-ray scattering (SAXS). Investigation of the SAXS curves normalized for concentration allows to detect the transition from the dilute to the semidilute regime. Some of the experimental curves exhibit a clear maximum. In the case of kappa-carrageenan, the position of this maximum is proportional to the cube root of polysaccharide concentration. The fluctuation theory of Borue and Erukhimovich (BE theory), which predicts a maximum in the scattering curves for low ionic strength, gives a good fit for all our experiments. An increase of the polymer concentration or the ionic strength causes the appearance of two subsystems in solution exhibiting a different characteristic screening scale of Coulombic interaction for ideal Gaussian chains (and two different segment lengths). The condition for an increase of the relative contribution of the subsystem with the larger segment length correlates with the one for the disorder-order transition of carrageenan. In the most concentrated (17.1 mg/ml) solution of Na(+)-iota-carrageenan, the increase of NaCl molarity above 0.06 M gives rise to additional scattered intensity at the smallest angles, indicating molecular association.

Carrageenan↗
PubMed1996

Influence of ionic effects on the ordering and association phenomena in dilute and semidilute carrageenan solutions.

A small angle X-ray scattering (SAXS) study was made on Na(+)-kappa- and Na(+)- or Li(+)-iota-carrageenan in aqueous solutions with and without added salt in the dilute (approximately 6 mg/ml) and semidilute (approximately 16 mg/ml) regime close to the critical concentration (approximately 9-11 mg/ml). This concentration should be understood to be determined by contacts between blobs rather than between isolated molecules. The SAXS data of carrageenan solutions are described in terms of the fluctuation model of Borue-Erukhimovich rather than by a particle-type model, although the latter form of scattering appears when aggregates of ordered fragments are formed. According to current models of carrageenan the ordered fragments are single or double chain helices. In conditions favorable for the appearance of the ordered conformation, two polymer subsystems with a different characteristic screening scale of Coulombic interaction for the ideal Gaussian chain are detected. The system with the larger characteristic screening scale corresponds to molecular fragments with an ordered conformation. The contribution of both systems to the scattering can be separated which allows for discussion of their behaviour in the framework of the fluctuation theory. The SAXS results suggest that in all dilute and in the semidilute solutions with added NaCl or LiCl for both kappa- and iota-carrageenans there is a transformation from short-length correlated (disordered) to longer-length correlated (ordered) molecular fragments followed by the association of the ordered fragments, whereas a transformation from an association of disordered fragments to a structure with ordered fragments is detected in semidilute solutions of Li(+)-iota-carrageenan in Li(+)-iota-carrageenan in LiI and Na(+)-kappa-carrageenan in NaI. In the semidilute solutions of Na(+)-iota-carrageenan in NaI the formation of ordered fragments seems to follow an intermediate sequence.

Carbohydrate Conformation↗
PubMed1996

X-ray diffraction study of in vitro calcification of tendon collagen.

Decalcified samples of turkey leg tendon were submitted to in vitro calcification in the presence of metastable solutions of calcium phosphate at different concentrations. The structural relationship between apatitic deposits and collagen fibrils was examined by high- and small-angle X-ray diffraction using conventional and synchrotron radiation sources. At high supersaturation the apatitic crystallites were deposited on the collagen fibrils with their crystallographic c-axis preferentially oriented parallel to the fibril axis. At lower supersaturation, a fraction of the apatitic crystallites also grew with the c-axis preferentially oriented parallel to the collagen fibril axis, whereas other exhibited a preferential orientation perpendicular to the fibril axis. The analysis of the small-angle X-ray diffraction data indicates that the deposition of the apatitic phase in the sample stored in solution at lower supersaturation induced modifications of the collagen electron density distribution in the axial direction, which can be attributed to the deposition of the inorganic crystallites inside the gap region of the collagen structure.

Animals↗
PubMed1996

Physicochemical characterization of protein-free low density lipoprotein models and influence of drug loading.

PURPOSE: Drug free and drug loaded protein-free low density lipoprotein (LDL) models consisting mainly of phospholipids, cholesterol, cholesterol esters, and triglycerides in ratios found for physiological LDL have been prepared. Their physicochemical characteristics were compared with those of physiological LDL. METHODS: Different characterization methods were used: photon correlation spectroscopy, transmission electron microscopy, X-ray solution scattering, and 1H nuclear magnetic resonance spectroscopy (NMR). RESULTS: Particle sizes are highly dependent on the preparation method and in particular on the homogenization conditions. Electron microscopy indicates that the size distributions of model systems are much broader than those of physiological LDL. The X-ray solution scattering patterns of the model systems display a temperature dependent maximum near 3.8 nm similar to that found in the patterns of physiological LDL. NMR indicates a comparable mobility of the lipid molecules in model particles and in physiological LDL. The influence of drug loading is similar to that found earlier for physiological LDL. In particular, the incorporation of the anti-cancer drug WB 4291 seems to have a fluidizing effect on the lipids in the core region of the particles. CONCLUSIONS: The preparation method of LDL model systems is of crucial importance as only the solvent evaporation method yielded systems in the size range of physiological LDL with acceptable high lipid concentrations. The fluidizing influence of temperature and drug incorporation (WB 4291) may be disadvantage in drug targeting.

Antineoplastic Agents↗
PubMed1996

Altered x-ray diffraction pattern is accompanied by a change in the mode of cross-link formation in lipodermatosclerosis.

We studied the molecular packing of collagen fibrils by x-ray diffraction in skin specimens of patients with lipodermatosclerosis and in controls. A difference in the tilt angles of the collagen molecules relative to the fiber axis is suggested by a D-stagger that is 1 nm larger in sclerotic skin than in normal skin. In parallel, the collagen cross-links in the skin specimens were analyzed, and a marked increase of both hydroxylysylpyridinoline and lysylpyridinoline, the trivalent mature cross-links characteristic of skeletal tissues, was found. The content of hydroxylysylpyridinoline and lysylpyridinoline was higher in the deep layer of the affected dermis than in the superficial dermis. This increase was always accompanied by an increase in the hydroxylysylpyridinoline/lysylpyridinoline ratio, suggesting that hydroxylysylpyridinoline is a sclerosis-associated cross-link. In addition, lysyl hydroxylation was increased in affected skin, and this increase was apparently restricted to the collagen telopeptides, which are crucial anchoring structures for lysyl dependent cross-links.

Amino Acids↗
PubMed1995

Nonideality of water-hexafluoropropanol mixtures as studied by X-ray small angle scattering.

Strong temperature dependent low angle X-ray scattering by trifluoroethanol- and hexafluoro-2-propanol-water mixtures was observed in conditions commonly used in NMR work on peptides. At least two types of molecular effects may contribute to the observed scattering: formation of clathrate hydrate-like aggregates of alcohol with water as have been proposed for the similar system tert-butanol-water (Iwasaki, K. and Fujiyama, T. (1976) J. Phys. Chem. 81, 1908-1912) and a further heterogeneity of the solution resulting from immiscibility of the two components.

1-Propanol↗
PubMed1995

Influence of phosphoenolpyruvate and magnesium ions on the quaternary structure of enzyme I of the phosphotransferase system from gram-positive bacteria.

Solution X-ray scattering patterns of enzyme I of the phosphotransferase system from Staphylococcus carnosus indicate an increase in radius of gyration and molecular mass in the presence of Mg2+ or both Mg2+ and phosphoenolpyruvate, indicating a partial dimerization of enzyme I. Mg2+ ions are essential for both the dimerization and the activation, whereas the substrate phosphoenolpyruvate shifts the monomer--dimer equilibrium to the enzymatically active dimer by decreasing the dissociation rate of the phosphorylated dimer.

Enzyme Activation↗
PubMed1995

Small-angle X-ray solution scattering study on the dimerization of the FKBP25mem from Legionella pneumophila.

The dimerization of the FK506-binding peptidyl-prolyl cis/trans-isomerase (PPIase) FKBP25mem (Mip (macrophage infectivity potentiator) protein) from Legionella pneumophila was studied by small-angle X-ray solution scattering. A value of 44 kDa, independent on the protein concentration between 2 and 13 mg/ml, confirming that FKBP25mem is a dimer was found for the molecular mass of the protein. The radius of gyration of the protein is 3.3 nm and the Porod volume 87 nm3. A model of the shape of FKBP25mem was evaluated from the scattering curve. Each monomer consists of a proximal and a peripheral domain, which are perpendicular to each other. The envelope of the crystallographic model of human FKBP12 fits well into the peripheral domain. The contact regions between the two monomers in the dimeric protein are probably located between the N-terminal parts of the monomers.

Amino Acid Isomerases↗
PubMed1995

Characterization of native and drug-loaded human low density lipoproteins.

Low-density lipoproteins (LDLs), the physiological vehicles for lipids, are potentially useful drug delivery devices for (hydrophobic) drugs. The physicochemical characteristics of LDL loaded with the adriamycin derivative AD 32 or the N-mustard derivative WB 4291 were compared to that of native and reconstituted LDL at different temperatures. X-ray solution scattering indicates that loading with AD 32 has no detectable effect on the particle structure at room temperature, in contrast to WB 4291. According to 19F NMR data, AD 32 molecules are located in two distinct chemical environments with restricted motional freedom of the CF3 groups in samples stored as lyophilisates. 1H NMR signals from AD 32 were not observed, while those from WB 4291 could be distinguished from those of LDL constituents. WB 4291 molecules are in an environment with a higher motional freedom than AD 32 molecules. 1H NMR data suggest a higher fluidity of the core components for the WB-loaded LDLs compared to the other LDL preparations. While the motional freedom of the phospholipid head groups seems to be temperature independent, there is an increase in the mobility of the lipid components in the core region of the LDL particles with temperature.

Calorimetry, Differential Scanning↗