Colin D. Reynolds

Post-translational modifications of the linker histone variants and their association with cell mechanisms

In recent years, a considerable amount of research has been focused on establishing the epigenetic mechanisms associated with DNA and the core histones. This effort is driven by the fact that epigenetics is intimately involved with genomics in a whole range of molecular processes. However, there is now a consensus that the epigenetics of the linker histones are just as important. The result of that consensus is that the post‐translational modifications (PTMs) for most of the linker histone variants in human and mouse have now been established by a number of experimental techniques, foremost of which is mass spectrometry (MS). MS was also used by our group to establish the PTMs of the linker histone variants in chicken erythrocytes. Although it is now known which types of PTM occur at particular locations on the linker histone variants, there is still a large gap in the knowledge of how this data relates to function. The focus of this review is an analysis of the PTM data for the linker histones from several species, but with an emphasis on human, mouse, and chicken. Our analysis reveals that certain PTMs can be clearly correlated with specific functions of the linker histones in particular cell types, and that unique PTM patterns exist for different cell types.

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.

Functionalised tetrathiafulvalene (TTF) systems derived from 4,5-(propylenedithio)-1,3-dithiole units

Abstract A range of functionalised symmetrical and unsymmetrical tetrathiafulvalene (TTF) derivatives containing substituted 4,5-(propylenedithio)-1,3-dithiole units has been prepared. Key half-units are the t-butyldiphenylsilyl-protected 1,3-dithiole derivative 18 and the ketal-protected derivative 32. Self-coupling and cross-coupling reactions of these half-units, with 1,3-dithiole-2-one and -2-thione derivatives 19–22 occurs in the presence of triethylphosphite. After deprotection, TTF derivatives 14, 24, 28, 34 and 40–43, bearing hydroxy or ketone functionality are obtained. Functionalisation of the alcohol group(s) of 14, 24 and 28 has been achieved with acid chlorides and with isocyanates, to give compounds 16, 17, 25, 26 and 29. Cyclic voltammetric studies establish that the new TTF derivatives are efficient π-electron donors; they undergo two reversible, single-electron redox waves. The X-ray crystal structures of 4,5-(2-hydroxypropylenedithio)-1,3-dithiole-2-thione 3 and the ketal-prote

Structure of the native (unligated) mannose-specific bulb lectin from Scilla campanulata (bluebell) at 1.7 Å resolution

The X-ray crystal structure of native Scilla campanulata agglutinin, a mannose-specific lectin from bluebell bulbs and a member of the Liliaceae family, has been determined by molecular replacement and refined to an R value of 0.186 at 1.7 A resolution. The lectin crystallizes in space group P21212 with unit-cell parameters a = 70. 42, b = 92.95, c = 46.64 A. The unit cell contains eight protein molecules of Mr = 13143 Da (119 amino-acid residues). The asymmetric unit comprises two chemically identical molecules, A and B, related by a non-crystallographic twofold axis perpendicular to c. This dimer further associates by crystallographic twofold symmetry to form a tetramer. The fold of the polypeptide backbone closely resembles that found in the lectins from Galanthus nivalis (snowdrop) and Hippeastrum (amaryllis) and contains a threefold symmetric beta-prism made up of three antiparallel four-stranded beta-sheets. Each of the four-stranded beta-sheets (I, II and III) possesses a potential saccharide-binding site containing conserved residues; however, site II has two mutations relative to sites I and III which may prevent ligation at this site. Our study provides the first accurate and detailed description of a native (unligated) structure from this superfamily of mannose-specific bulb lectins and will allow comparisons with a number of lectin-saccharide complexes which have already been determined or are currently under investigation.

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 Å and 1.35 Å, respectively. In both crystal forms, the SNA‐II molecule folds into two linked ββ‐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, αα1‐methylgalactose, fucose, and the carcinoma‐specific Tn antigen) were determined at 1.55 Å 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. Proteins 2009.

Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin

The three‐dimensional structure of a 244‐residue, multivalent, fetuin‐binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 Å resolution by molecular replacement using the coordinates of the 119‐residue, mannose‐binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose‐binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12‐stranded β‐prism II fold, with three putative carbohydrate‐binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.

X-ray structure solution of amaryllis lectin by molecular replacement with only 4% of the total diffracting matter.

It is often the case that analogous proteins from different species crystallize in a different form. These structures can usually be easily solved by the molecular-replacement (MR) technique, as the protein folding is very often conserved. However, the results from MR become more uncertain as the proportion of diffracting matter decreases as a result of multimericity and/or absence of some of the atoms in the model. In this paper results are presented on the structure solution of amaryllis lectin (109 residues per monomer) containing two protein molecules in the asymmetric unit. The structure was solved by MR using the Calpha coordinates of one monomer from snowdrop lectin which has 85% amino-acid sequence identity to amaryllis lectin. This represents only 6% of the non-H atoms of the protein molecule to be used for structure determination and it is a major improvement on previous reports. Further calculations were carried out in order to establish the minimum number of atoms which could be included in the model before a clear solution to the MR problem was revealed. This study showed that the structure of amaryllis lectin could still have been solved easily with 3.85% of the model, which even in the most favourable cases, will probably constitute a minimum for molecular-replacement structure solution.

Purification, crystallization and preliminary X-ray analysis of a mannose-binding lectin from bluebell (Scilla campanulata) bulbs.

Crystals have been grown of a mannose-specific lectin from bluebell (Scilla campanulata) bulbs in a form suitable for X-ray diffraction studies. The crystals, which diffract to high resolution, grew in hanging drops by vapour diffusion, equilibrating with a solution of 70% saturated ammonium sulfate at pH 4.7-4.8 at 293 K, in the absence of any mannose saccharides. Crystals are orthorhombic, P2(1)2(1)2, with unit-cell dimensions a = 70.78, b = 93.69, c = 46.92 A. The functional lectin molecule is organized as a tetramer of four identical 14 kDa subunits, with only two subunits in the asymmetric unit. Data to 1.86 A resolution have been recorded and the structure determined by the molecular replacement method.

Histone Structures: Targets for Modifications by Molecular Assemblies

Abstract: 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 α‐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.

Crystal structure of monomeric hydrated iron(III) 5,10,15,20-tetra(N-methyl-4-pyridinio)porphyrin pentachloride; an ionic haem model

The crystal structure of hydrated iron(III) 5,10,15,20-tetra(N-methyl-4-pyridinio)porphyrin pentachloride has been determined. It crystallises as high-spin six-co-ordinated diaqua monomers (molecular symmetry ). The iron atom lies in the plane of a flat porphyrin ring [Fe–N(porphyrin), 2.042(6) and 2.038(7)A]. The axial water ligands [Fe–H2O 2.086(5)A] are hydrogen-bonded to chloride ions and water molecules in a network of negative charges that interlace between the positively charged iron(III) porphyrins.