Structure of histone octamers in reconstituted polynucleosomes

The salt-dependent structural changes of the histone octamer in complex with high-molecular-weight DNA have been studied by fluorescent spectroscopy. Changes in both the spectra maximum position and anisotropy of the histone tyrosine fluorescence reveal structural transitions in nucleosome within the ranges of 0.5-3 mM and 20-30 mM NaCl. Comparison of the octamer fluorescent parameters in complex with DNA as well as in a free state permits to interpret the revealed structural transitions as a change in degree of contacts stability between (H2A-H2B) dimer and (H3-H4)2 tetramer. More pronounced conformational changes in histone octamer are observed under the conditions of polynucleosome fibers interaction within the range of physiological ionic strength (100-600 mM NaCl). As far as fluorescent parameters are concerned, the aforementioned changes are connected with entire destruction of (H2A-H2B) dimer specific contacts with (H3-H4)2 tetramer. The obtained results suggest the possibility of existence of different structural states of histone octamer in the chromatin composition including those which are quite dissimilar from the octamer structure in the 2M NaCl solution.     

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4α and 4β subunits arranged in a dihedral point group symmetry D₂. The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0° tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.     

The first three-dimensional structure of phosphofructokinase from Saccharomyces cerevisiae determined by electron microscopy of single particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4alpha and 4beta subunits arranged in a dihedral point group symmetry D(2). The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0 degrees tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.     

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.     

Influence of secondary structure of polyglycine on some conformational properties

Values of four conformational properties, namely unperturbed dimension [r2]0, dipole moment [mu 2], mean squared optical anisotropy [gamma 2], and molar Kerr constant [mK], have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, alpha-helix or beta-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations alpha-helix/beta-sheet and beta-sheet/random coil were allowed and the results obtained compared with previous work for alpha-helix/random coil chains.     

Spectropolarimetric analysis of the core histone octamer and its subunits

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.     

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa.

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.     

An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.     

Interaction of alpha-gliadin with poly(HEMA-co-SS): structural characterization and biological implication

The wheat gluten protein alpha-gliadin, a well known trigger of celiac disease, can be complexed by random copolymers of hydroxyethyl methacrylate (HEMA) and sodium 4-styrene sulfonate (SS). In this work, influence of alpha-gliadin and poly(HEMA-co-SS) concentrations on alpha-gliadin structure was studied using spectroscopic techniques and dynamic light scattering. In 70% ethanol or 0.06M HCl (pH 1.2), alpha-gliadin was found to self-associate upon increasing its concentrations and displayed decreased alpha-helical content and increased beta-turn and beta-sheet contents. At pH 1.2, alpha-gliadin interacted with poly(HEMA-co-SS) to form supra-molecular complex particles. Poly(HEMA-co-SS) induced alpha-gliadin structural changes that mimicked those obtained by varying the protein concentration in pure solution. At pH 6.8, alpha-gliadin was poorly soluble and formed large particles but alpha-helix is still main secondary structure. The influence of the polymer on protein structure was weaker at neutral than acidic pH. Interaction with poly(HEMA-co-SS) disrupted alpha-gliadin conformation and self-association to form new complex particles at neutral pH. This study provides insight into the mechanism of poly(HEMA-co-SS)/alpha-gliadin interaction and the polymer as alpha-gliadin sequestering agents in the supportive treatment of celiac disease.     

Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity

The far-ultraviolet circular dichroism (CD) spectra of the extracellular portion (papain-cleaved fragment) of the histocompatibility antigen H-2Kb and its noncovalently associated components, heavy chain and beta 2-microglobulin (beta 2m), indicate that the antigen is highly structured, containing about 30% alpha-helix, 41% beta-sheet, and 29% random coil. Separation of beta 2m from the heavy chain produced a decrease in heavy chain alpha-helix and beta-sheet structure which correlated with a loss of alloantigenic reactivity. Reconstitution of the heavy chain-beta 2m complex resulted in an increase in secondary structure which was greater than the sum of the free chains and the recovery of considerable alloantigenic reactivity. This suggests that some of the secondary structure and much of the alloantigenic reactivity may depend on conformation associated with the binding of beta 2m to heavy chain. A prediction of heavy chain secondary structure based on Chou-Fasman analysis of the primary amino acid sequence agreed with results from CD measurements and suggested that the segments of alpha-helix and beta-sheet structure are distributed throughout the molecule.     

A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).     

The oxidised histone octamer does not form a H3 disulphide bond

A H3 dimer band is produced when purified native histone octamers are run on an SDS-PAGE gel in a beta-mercaptoethanol-free environment. To investigate this, native histone octamer crystals, derived from chicken erythrocytes, and of structure (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A'), were grown in 2 M KCl, 1.35 M potassium phosphates and 250-350 microM of the oxidising agent S-nitrosoglutathione, pH 6.9. X-ray diffraction data were acquired to 2.10 A resolution, yielding a structure with an Rwork value of 18.6% and an Rfree of 22.5%. The space group is P6(5), the asymmetric unit of which contains one complete octamer. Compared to the 1.90 A resolution, unoxidised native histone octamer structure, the crystals show a reduction of 2.5% in the c-axis of the unit cell, and free-energy calculations reveal that the H3-H3' dimer interface in the latter has become thermodynamically stable, in contrast to the former. Although the inter-sulphur distance of the two H3 cysteines in the oxidised native histone octamer has reduced to 6 A from the 7 A of the unoxidised form, analysis of the hydrogen bonds that constitute the (H4-H3)-(H3'-H4') tetramer indicates that the formation of a disulphide bond in the H3-H3' dimer interface is incompatible with stable tetramer formation. The biochemical and biophysical evidence, taken as a whole, is indicative of crystals that have a stable H3-H3' dimer interface, possibly extending to the interface within an isolated H3-H3' dimer, observed in SDS-PAGE gels.     

Control of antibody-antigen interaction using anion-induced conformational change in antigen peptide

The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions. We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)n and (LLKK)n. In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel alpha-helical structure and the epitope was subsequently 'bent'. In the presence of 41 microM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5. Oligo-DNAs, as anions, dissociated the antibody-peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody-peptide complex at pH 7.5 were 4x10(-8), 1x10(-7) and 6x10(-6) M for decamer, octamer and hexamer DNA, respectively.     

Alpha-helix folding by Monte Carlo simulated annealing in isolated C-peptide of ribonuclease A

Conformation of the C-peptide fragment of RNase A is calculated by Monte Carlo simulated annealing. We adopt the total potential energy as given by the sum of generic interatomic energies whose parameters are determined separately for each amino acid without referring to the empirical structure of the C-peptide. The simulation is carried out in a completely unrestricted way without imposing any weight towards given final destinations. Starting from completely random initial conformations and minimizing the total potential energy with respect to main-chain dihedral angles and side-chain torsion angles, we have obtained partial alpha-helix structure with a high probability (approximately 40%). The energetically most favourable structure exhibits a 2.5-turn alpha-helix at the location identical with that of the 3-turn alpha-helix in the native enzyme molecule. Classification of conformations obtained in the simulation into clusters of similar structure shows that our simulation indeed predicts the alpha-helix structure for the isolated C-peptide with specific charged residues. The results of simulation with various amino acid substitutions are also found to be consistent with the experimental implication for the importance of intramolecular ionic interactions for alpha-helix stability for this peptide.     

Evidence for an alpha-helix --> pi-bulge helicity modulation for the neu/erbB-2 membrane-spanning segment. A 1H NMR and circular dichroism study

The 35-residue peptide corresponding to the very hydrophobic transmembrane region of the tyrosine kinase receptor neu, Neu(TM35), has been synthesized. The peptide can be solubilized in millimolar concentrations in TFE or incorporated into an SDS-water micellar solution or into well-hydrated DMPC/DCPC bicelles. In all these media, circular dichroism demonstrated that the peptide adopts a helical structure for about 80% of its amino acids. The peptide is monomeric below 2 mM in TFE, as also determined by variable concentration experiments. The three-dimensional solution structure in TFE has been obtained by homonuclear proton NMR and shows a well-defined alpha-helix from residues 4 to 21, then a pi-bulge from Ile(22) to Gly(28), and a final short alpha-helix from positions 29 to 32. This experimental finding is in agreement with structures predicted recently by molecular dynamics calculations in a vacuum [Sajot, N., and Genest, M. (2000) Eur. Biophys. J. 28, 648-662]. The biological implications of a possible retention of this structure in a membrane environment are finally discussed.     

Capillary electrophoresis is a sensitive monitor of the hairpin-random coil transition in DNA oligomers

Capillary electrophoresis (CE) has been used to characterize the hairpin-random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 degrees C. The TTTT octamer forms a minihairpin with a melting temperature of 29 degrees C, the TTTC octamer has a melting temperature of 16 degrees C, and the AAAA octamer has a melting temperature below 0 degrees C. The thermal transitions of the TTTT, TTTC, and AAAA octamers are well fitted by a structure prediction algorithm; however, the GAAA minihairpin is considerably more stable than predicted. The melting temperature of the GAAA minihairpin is reduced to 47 degrees C in aqueous buffers containing 7.2M urea and to 33 degrees C in buffers containing 7.2M urea plus 40% (v/v) formamide. The combined results indicate that CE is a sensitive technique for monitoring conformational transitions in small DNA molecules.     

甘蓝型油菜β碳酸酐酶的结构预测

根据已经克隆到的甘蓝型油菜β碳酸酐酶基因序列,概念地翻译成蛋白质的氨基酸序列。利用Vector NTISuite、SOPMA、Swiss-Model和NCBI-VAST等软件和服务器对甘蓝型油菜β碳酸酐酶的一级结构、二级结构、三维结构进行分子结构模型预测,并进行三维结构的比对。预测结果显示,甘蓝型油菜β碳酸酐酶是定位于叶绿体基质的蛋白质,具有β类碳酸酐酶所特有的保守性基序Cys-Xn-His-X2-Cys;SOPMA预测二级结构显示α螺旋(39.88%)、随机卷曲(39.27%)、β折叠(16.31%)和β转角(4.53%);用同源建模法构建了三维结构图;通过VAST矢量比对工具将甘蓝型油菜β碳酸酐酶与模板(1ekjG)进行三维结构比对,显示甘蓝型油菜β碳酸酐酶与豌豆β碳酸酐酶同型八聚体中的一个单体(1ekjG)很好的匹配,推测甘蓝型油菜β碳酸酐酶全酶也是同型八聚体。     

Interaction of cisplatin drug with Na,K-ATPase: drug binding mode and protein secondary structure

cis-Pt(NH(3))(2)Cl(2) (cisplatin) is an antitumor drug with many severe toxic side effects including enzymatic changes associated with its mechanism of action. This study was designed to examine the interaction of cisplatin drug with the Na(+), K(+)-dependent adenosine triphosphatase (Na,K-ATPase) in H(2)O and D(2)O solutions at physiological pH, using drug concentrations of 0.1 microM to 1 mM. UV absorption spectra and Fourier transform infrared difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the drug binding mode, the drug binding constant and the protein secondary structure in the cisplatin-ATPase complexes. Spectroscopic evidence showed that at low drug concentration (0.1 microM), cisplatin binds mainly to the lipid portion of the enzyme, whereas at higher drug contents, the Pt cation interaction is through the polypeptide C==O and C-N groups with overall binding constant of K=1.93 x 10(4) M(-1). At high cisplatin concentration (1 mM), drug binding results in protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the alpha-helix 22.2%; beta-pleated 23.2%; turn 9.4%; beta-antiparallel 2.2% and random 43%, in the cis-Pt-ATPase complexes.     

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.