Cofractionation of HMGB proteins with histone dimers.

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A-H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein-protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.

Alpha-glucosidase inhibitor proteins from Sesbania grandiflora flowers.

Two alpha-glucosidase inhibitors were isolated from the flowers of Sesbania grandiflora and named SGF60 and SGF90. The procedure involved extraction with phosphate buffer, precipitation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose and gel filtration on Superdex-200. These proteins were identified by using tandem mass spectrometry. The results show partial amino acid sequences of SGF60 similar to p27SJ, a protein from Hypericum perforatum found to suppress HIV-1 gene expression. SGF90 matched a beta-glucosidase from Arabidopsis thaliana.

Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm.

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.

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.

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from the edible mushroom Schizophyllum commune.

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.

A bacteriophytochrome regulates the synthesis of LH4 complexes in Rhodopseudomonas palustris.

The non-sulphur purple bacterium Rhodopseudomonas palustris contains five pucAB genes for peripheral light-harvesting complexes. Bacteria grown under high-light conditions absorb at 800 and 850 nm but in low-light the 850 nm peak is almost absent and LH2 complexes are replaced by LH4. The genome contains six bacteriophytochromes (Bph). Bphs sense light in the red/far-red through a reversible Pr to Pfr transformation that controls gene expression. Bph3 (RPA1537) controls the expression of a cluster of photosynthetic genes, however most of the peripheral light harvesting complex genes are outside of this region. The pucAB-d genes encode LH4 peptides and are near two Bphs (RPA3015, RPA3016). We have characterised three Bphs and show that Bph4 RPA3015 and Bph3 RPA1537 have different dark stable states. It is known that Bph3 is active in its red absorbing Pr form and suggests a working hypothesis that Bph4 is active in the Pfr state. We show that LH4 expression can be induced with red light at the Pr absorption maximum (708 nm) of Bph4. The property of light transmission of water maybe an important factor in understanding this adaptation. Bph4 can sense the reduction in light intensity indirectly through an increase in ratio of transmitted red/far-red light. The red right activated Bph4 regulates the synthesis of LH4 which concentrates bacteriochlorophyll a pigment absorption at 800 nm to exploit a recovery in water light transmission in this region.

High-resolution structure of the native histone octamer.

Crystals of native histone octamers (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A') from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 A resolution, yielding a structure with an R(work) value of 18.7% and an Rfree of 22.2%. The crystal space group is P6(5), the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A'-H3-H4 molecular cluster (also H2A-H3'-H4'), the H4-H2B' interaction (also H4'-H2B) and the H2A'-H4 beta-sheet interaction (also H2A-H4'). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.

Histone structures: targets for modifications by molecular assemblies.

The core histone proteins contain modification sites that are key elements in the regulation of the cell cycle, DNA replication and repair with histone assembly, control of gene expression, and transcriptional elongation. Much work has been done on the various molecular assemblies that remodel nucleosomes, methylate, ubiquitinate, and cause ADP-ribosylation of histones, and acetylate and phosphorylate core histone tails. The core histones are the final targets of the enzymes in the molecular assemblies. What structural changes in the histones are correlated with these modifications? This paper considers the high-resolution structure of the histone octamer and stresses the importance of histone docking sequences in the binding of the two (H2A-H2B) dimers to the (H3-H4)(2) tetramer. There is an extensive acid-base area of interaction between histone octamers in crystals at high salt, which may have implications for nucleosome remodeling. We show that there are regions of high alpha-helix probability in all core histone N-terminal tails in regions where lysine acetylation occurs. There are also consensus sequences spanning up to eight amino acid residues between some histone tail regions. Circular dichroism studies using synchrotron radiation at wavelengths as low as 130 nm are promising for the accurate measurement of changes of histone secondary structure related to function.