Crystallization and preliminary X-ray investigation of a recombinant form of human gamma-interferon

A genetically engineered human gamma-interferon has been crystallized from solutions of ammonium sulfate. The crystals are trigonal, space group R32, with hexagonal axes of a = 114.0(1) A and c = 314.9(2) A. The crystals are moderately stable to x-rays and diffract to 2.85-A resolution. Density measurements indicate that the crystallographic asymmetric volume contains four interferon molecules.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.     

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.     

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.     

Crystals of a toxic protein from the venom of the scorpion Centruroides sculpturatus Ewing: preparation and preliminary x-ray investigation

A toxic protein from the venom of the scorpion Centruroides sculpturatus has been crystallized. The crystals are orthorhombic, with unit cell dimensions $\text{a = 52.2}\text{A}\text{?}\text{, b = 42.0}\text{A}\text{?}\text{and}\text{c = 28.5}\text{A}\text{?}$, space group P2₁2₁2₁. Density measurements indicate that there are four molecules of toxin in the unit cell.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.     

Fibroblast biology: Development and differentiation of synovial fibroblasts in arthritis

Synovial fibroblasts occur as two phenotypes - intimal and subintimal. The specialised intimal phenotype includes expression of uridine diphosphoglucose dehydrogenase (UDPGD), vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF). These gene products contribute to specialised functions relating to tissue movement and leucocyte traffic.