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Z Sayers

Publications and source records attributed to Z Sayers.

18 recordsLinked to original sources
PubMed2003

Structural characterization of T-protein of the Escherichia coli glycine cleavage system by X-ray small angle scattering.

T-protein, one of the components of the glycine cleavage complex, catalyses the formation of ammonia and methylene-tetrahydrofolate from H-protein-bound intermediate. Native T-protein of the glycine cleavage system from E. coli was efficiently purified using a combination of hydrophobic interaction, gel permeation and ion exchange chromatography. Synchrotron radiation small angle X-ray solution scattering indicates that T-protein has an extended structure in solution. A low resolution model of the protein was constructed ab initio and tentative models of the tertiary structure were built using prediction methods constrained by the scattering data.

Aminomethyltransferase↗
PubMed1999

Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.

Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].

Carrier Proteins↗
PubMed1998

Interactions of elongation factor 2 with the cytoskeleton and interference with DNase I binding to actin.

Interactions of elongation factor 2 (EF-2) with G-actin and F-actin in vitro were investigated using viscosimetry, gel filtration and electron microscopy. Under depolymerization conditions, at a molar ratio of 0.5:1 (EF-2/F-actin subunit), F-actin is stabilised by EF-2 and filaments depolymerize about three times slower than control solutions containing only F-actin. Filament stability is improved also when EF-2 is included in the solution in the presence of DNase I. Electron micrographs and viscosity measurements indicate that EF-2 may support small bundles with a width of 2 or 3 filaments. It was established that EF-2 interacts with G-actin in vitro, and reduces G-actin inhibition of DNase I activity when it is present at a ratio of 1:1. Results are discussed in the context of possible functional significance of the interactions.

Actins↗
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

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↗
PubMed1994

Interactions of eukaryotic elongation factor 2 with actin: a possible link between protein synthetic machinery and cytoskeleton.

Eukaryotic elongation factor 2 (EF-2) was shown to bind to F-actin as assayed by co-sedimentation. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding was increased fourfold. At saturation level a molar ratio of about 0.12 EF-2 per F-actin (subunit) was observed. Our results suggest a single type of binding site with an apparent dissociation constant of 0.85 microM. The stoichiometry was independent of the filament length, and ADP-ribosylation had no effect on the binding. Experimental data indicated the involvement of SH-groups of both EF-2 and actin in the binding. The interaction EF-2 with F-actin appeared to be inhibited competitively by EF-1 alpha and non-competitively by G-actin.

Actins↗
PubMed1993

Cloning and expression of Saccharomyces cerevisiae copper-metallothionein gene in Escherichia coli and characterization of the recombinant protein.

The gene sequences for intact and truncated forms of copper-binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells. In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+. The recombinant truncated protein binds copper in vivo and in vitro. A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X-ray fluorescence and amino acid analysis. Extended X-ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms.

Base Sequence↗
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

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↗
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↗
PubMed1985

Time-resolved X-ray scattering study of actin polymerization from profilactin.

The polymerization of actin in solutions of purified calf spleen actin or profilactin (1-10 mg . ml-1) was followed by synchrotron radiation X-ray solution scattering. At the concentration used, polymerization of actin from profilactin or actin occurs without any lag phase. It is shown by a combination of solution scattering, model calculations and electron microscopy that contrary to the conclusions from previous viscometry studies, filaments form without any lag phase in profilactin solution but aggregate in bundles or networks. This phenomenon is independent of the method used to induce polymerization: slow temperature increase, temperature jump in the presence of polymerizing salts or fast mixing with salt. This aggregation explains the lower final viscosity levels, as compared to actin solutions, observed during the polymerization of actin from profilactin.

Actins↗
PubMed1981

The distribution of electron density in corneal collagen fibrils.

Using high intensity synchrotron X-ray diffraction data, an electron density profile has been obtained for the collagen fibrils in corneal stroma at higher resolution than has been possible before. The details within this electron density distribution are discussed in terms of the collagen and its interactions with other components present in the tissue.

Collagen↗