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Biomedical subjects

M H Koch

Publications and source records attributed to M H Koch.

At least 19 recordsLinked to original sources
PubMed1998

Biophysical characterisation of lysozyme binding to LPS Re and lipid A.

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The beta<-->alpha gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stoichiometry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly alpha helix to more unordered structures takes place, while the residual beta-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.

Calorimetry↗
PubMed1998

The charge of endotoxin molecules influences their conformation and IL-6-inducing capacity.

The activation of cells by endotoxin (LPS) is one of the early host responses to infections with Gram-negative bacteria. The lipid A part of LPS molecules is known to represent the endotoxic principle; however, the specific requirements for the expression of biologic activity are still not fully understood. We previously found that a specific molecular conformation (endotoxic conformation) is a prerequisite for lipid A to be biologically active. In this study, we have investigated the interdependence of molecular charge and conformation of natural and chemically modified LPS and lipid A and its transport and intercalation into phospholipid membranes mediated by human LPS-binding protein, as well as IL-6 production after stimulation of whole blood or PBMCs. We found that the number, nature, and location of negative charges strongly modulate the molecular conformation of endotoxin. In addition, the LPS-binding protein-mediated transport of LPS into phospholipid membranes depends on the presence of net negative charge, yet charge is only a necessary, but not a sufficient, prerequisite for transport and intercalation. The biologic activity is determined mainly by the molecular conformation: only conical molecules are highly biologically active, whereas cylindrical ones are largely inactive. We could demonstrate that the net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity.

Acute-Phase Proteins↗
PubMed1998

Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding.

Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover. This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences. It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein. Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state. The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little. Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein. Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an alpha-beta-barrel using discrete state-space modelling.

Circular Dichroism↗
PubMed1998

Escherichia coli SecA shape and dimensions.

SecA shape and conformational flexibility in solution were studied by small angle X-ray scattering. Dimeric SecA is a very elongated molecule, 15 nm long and 8 nm wide. SecA is therefore four times as long as the membrane is wide. The two globular protomers are distinctly separated and share limited surface of intermolecular contacts. ATP, ADP or adenylyl-imidodiphosphate (AMP-PNP) binding does not alter the SecA radius of gyration. A SecA mutant that catalyzes multiple rounds of ATP hydrolysis does not undergo conformational changes detectable by small angle X-ray scattering (SAXS). We conclude that SecA conformational alterations observed biochemically during nucleotide interaction are only small-scale and localized. The ramifications of these findings on SecA/SecYEG interaction are discussed.

Adenosine Diphosphate↗
PubMed1998

Solution structure of the ternary complex between aminoacyl-tRNA, elongation factor Tu, and guanosine triphosphate.

Complex formation between elongation factor Tu (EF-Tu), Phe-tRNAPhe, and GTP was analyzed by small-angle neutron and X-ray scattering methods. Both techniques show that the ternary complex consists of one EF-Tu and one aminoacyl-tRNA. No shift in stoichiometry was detected when the temperature was raised from 5 to 37 degreesC, in contrast to previous observations obtained from RNase A protection experiments [Bilgin and Ehrenberg (1995) Biochemistry34, 715-719]. A small but significant increase in the radius of gyration of the complex was observed when the temperature was decreased from 37 to 5 degreesC. The X-ray solution scattering patterns were compared with those calculated from the crystal structure of the complex formed between EF-Tu from Thermus aquaticus and Phe-tRNAPhe from yeast. The comparison shows that the solution structure of the ternary complex, formed entirely from Escherichia coli components and under translationally optimal buffer conditions, is very close to the crystal structure, formed from heterologous components under very different conditions. Furthermore, for the hybrid complex in solution there is no evidence for the formation of trimers as suggested by the crystal structure.

Binding Sites↗
PubMed1998

Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA).

The enzyme UDP-N-acetylglucosamine (UDP-GlcNAc) enolpyruvyltransferase (MurA), the target of the antibiotic fosfomycin, was investigated by small-angle X-ray scattering (SAXS) and fluorescence spectroscopy to detect conformational changes that had been proposed on the basis of the crystal structure of unliganded and liganded MurA. The SAXS data indicate that binding of UDP-GlcNAc to free enzyme results in substantial conformational changes, which can be interpreted as the transition from an open to a closed form. Fosfomycin did not affect the structure of free enzyme or sugar-nucleotide-bound MurA. Phosphoenolpyruvate (pyruvate-P) appeared to induce a structural change upon addition to free enzyme, which differed from that observed upon binding of UDP-GlcNAc. Fluorescence experiments were performed using the hydrophobic fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS). The fluorescence quenching of MurA/ANS solutions upon addition of UDP-GlcNAc or pyruvate-P was concentration dependent in a saturating manner, yielding apparent dissociation constants of K(d(UDP-GlCNAc)) = 59 microM and K(d(pyruvate-P)) = 240 microM. The results suggest that binding of substrates does not exclusively follow an ordered mechanism with UDP-GlcNAc binding first, although binding of UDP-GlcNAc to free enzyme is preferred and possibly influenced by pyruvate-P. The reaction thus appears to follow an induced-fit mechanism, in which the binding site for fosfomycin, and presumably also for pyruvate-P, is created by the interaction of free enzyme with the sugar nucleotide. The methods described here provide a tool for the characterization of site-directed mutants of MurA and the interaction of this enzyme with potential inhibitors.

Alkyl and Aryl Transferases↗
PubMed1998

Small-angle X-ray solution-scattering studies on ligand-induced subunit interactions of the thiamine diphosphate dependent enzyme pyruvate decarboxylase from different organisms.

The quaternary structures of the thiamine diphosphate dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from the recombinant wild type of Saccharomycescerevisiae and Zymomonas mobilis and from germinating Pisum sativum seeds were examined by X-ray solution scattering. The dependence of the subunit association equilibrium on the pH and the presence of the cofactors thiamine diphosphate and magnesium ions were compared, and the differences between the catalytic properties of the different enzymes are discussed. The influence of amino acid substitutions at the cofactor binding site of the enzyme from Saccharomyces cerevisiae (E51 is substituted by Q or A and G413 by W) on the subunit association was examined. Low-resolution models of the P. sativum, Z.mobilis, and S. cerevisiae enzymes were evaluated ab initio from the scattering data. The enzyme from the bacterium and yeast appear as a dimer of dimers, whereas the plant enzyme is an octamer formed by two tetramers arranged side-by-side. The shape of the S. cerevisiae enzyme agrees well with the atomic structure in the crystal but suggests that the dimers in the latter should be tilted by approximately 10 degrees. The resulting modification of the atomic structure also yields a significantly better fit to the experimental solution scattering data than that calculated form the original crystallographic model.

Bacterial Proteins↗
PubMed1998

Protein hydration in solution: experimental observation by x-ray and neutron scattering.

The structure of the protein-solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density approximately 10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.

Escherichia coli↗
PubMed1998

X-ray diffraction and polarizing optical microscopy investigation of the structural organization of rabbit tibia.

X-ray diffraction and polarized optical microscopy investigations were carried out on thin sections of rabbit tibia in order to study the morphological organization of the structural components of this tissue, which often is utilized to test bone response to implants. In the optical microscope, the lateral face as well as the lateral portion of the caudal face exhibit a lamellar structure with an alternation of dark and bright lamellae running parallel to the long axis of the tibia. In contrast, both in the medial face and in the medial portion of the caudal face there are numerous osteonic structures. In spite of the complexity of this morphological organization, the results of small- and high-angle X-ray diffraction analyses indicate that the structural relationship between collagen fibrils and inorganic crystals is quite similar to that observed in single osteons and allows evaluation of the orientation of the two main structural components. Both collagen fibrils and apatitic crystallites are preferentially oriented parallel to the long axis of the tibia. The degree of orientation is greater in the thickness than in the plane of the lamellae, suggesting that collagen fibrils and inorganic crystallites lie preferentially in the plane of the lamellae, where they follow an oblique course. The degree of orientation of the apatitic crystallites is higher in the lateral face than in the medial and caudal faces, in agreement with the optical microscopic images. The results provide information that must be taken into account when evaluating the structural modifications of bone due to the insertion of a prosthetic device.

Animals↗
PubMed1998

X-ray diffraction on cyclically loaded osteons.

The results of a study on the fine structural distortion due to the two previously observed types of degradation in cyclically loaded single osteons (i.e., stiffness degradation and pinching effect) are presented. Fully calcified longitudinal and alternate osteons were isolated from 350-microns-thick longitudinal sections of human femoral cortical bone. The samples were prepared from 500-microns-long central cylindrical portions of an osteon, whose two ends were penetrating into rectangular lugs for fixation to an electromechanical device that cyclically loaded the samples. This device was connected to a microwave micrometer and a recorder. The structural distortions induced by cyclic loading were investigated by high- and low-angle X-ray diffraction on conventional and synchrotron radiation sources. Cyclic loading results in a reduction in the degree of orientation of apatite crystallites, especially in longitudinal osteons, in which the most abundant longitudinal lamellae are not protected against buckling by transverse lamellae as they are in alternate osteons. In contrast, the degree of orientation of collagen fibrils does not seem to be affected by cycling loading in the two osteon types, possibly because the disorientation of collagen fibrils is, within limits, a reversible process. Finally, the contrast between the disorientation of inorganic crystallites and the apparently unaltered distribution of collagen fibrils suggests that the degradation of cyclically loaded osteons may be due to a separation of the crystallites from the fibrils.

Adult↗
PubMed1998

Evidence for charge-controlled conformational changes in the photocycle of bacteriorhodopsin.

The existence of two different M-state structures in the photocycle of the bacteriorhodopsin mutant ASP38ARG was proved. At pH 6.7 (0 to -6 degreesC) a spectroscopic M intermediate (M1) that does not differ significantly in its tertiary structure from the light-adapted ground state accumulates under illumination. At pH > 9 another state (M2), characterized by additional pronounced changes in the Fourier transform infrared difference spectrum in the region of the amide I and II bands, accumulates. The M2 intermediate trapped at pH 9.6 displays the same changes in the x-ray diffraction intensities under continuous illumination as previously described for x-ray experiments with the mutant ASP96ASN. These observations indicate that in this mutant the altered charge distribution at neutral pH controls the tertiary structural changes that seem to be necessary for proton translocation.

Bacteriorhodopsins↗
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↗