PubMed Health⌕ Search

Biomedical subjects

C D Reynolds

Publications and source records attributed to C D Reynolds.

At least 19 recordsLinked to original sources
PubMed2003

Structure of the histone-core octamer in KCl/phosphate crystals at 2.15 A resolution.

The structure of the native chicken histone octamer, crystallized in 2 M KCl, 1.35 M potassium phosphate pH 6.9, has been refined at 2.15 A resolution to a final R factor of 21.4% and an R(free) of 25.2%. Unique crystal-packing interactions between histone-core octamers are strong and one of them (area 4000 A(2)) involves two chloride ions and direct interactions between six acidic amino-acid residues on one octamer and the equivalent number of basic residues on the next. These interactions are on the structured part of the octamer (not involving tails). Five phosphate ions, 23 chloride ions and 437 water molecules have been identified in the structure. The phosphate and some chloride ions bind to basic amino-acid residues that interact with DNA in the nucleosome. The binding of most of the anions and the packing interactions are unique to these crystals. In other respects, and including the positions of four chloride ions, the octamer structure is very close to that of octamers in nucleosome-core particle crystals, particularly with respect to 'docking' sequences of the histone H2As and H4s. These sequences together with the H2B-H4 four-helix bundles stabilize the histone structure in the nucleosome and prevent the dissociation of the (H2A-H2B) dimers from the (H3-H4)(2) tetramer. Possible reasons why this happens at high salt in the absence of DNA are given.

Amino Acid Sequence↗
PubMed2000

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 A 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 beta-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.

Amino Acid Sequence↗
PubMed1999

Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.

Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %. The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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

Amino Acid Sequence↗
PubMed1999

Crystallization of Helix pomatia agglutinin (HPA), a protein from the edible snail.

Crystals of Helix pomatia agglutinin (HPA) have been grown by the hanging-drop technique using polyethylene glycol as the precipitant at 293 K. Over a period of one to two weeks the crystals grew to maximum dimensions of 0.10 x 0.05 x 0.02 mm. The crystals belong to space group P6(3)22, with unit-cell dimensions a = b = 63.3, c = 105. 2 A and Z = 12 identical monomers of M(r) = 13 kDa, aggregating into two 78 kDa hexameric protein molecules per unit cell, each with symmetry 32 (D(3)). The diffraction pattern extends to 3.6 A at 293 K.

Animals↗
PubMed1998

Scilla campanulata agglutinin crystallized in complex with the trimannoside alpha-D-man-(1-->6)-[alpha-D-man-(1-->3)]-alpha-D-Man.

The monocot mannose-specific lectin, Scilla campanulata agglutinin (SCA), from bluebell bulbs has a strong affinity for alpha1,3- and alpha1,6-linked mannosyl residues. SCA has been co-crystallized with the trisaccharide alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->3)-alpha-D -mannopyranoside ¿alpha-D-Man-(1-->6)-[alpha-D-Man-(1-->3)]-alpha-D-Man¿, the core structure of biantennary N-linked oligosaccharides. Crystals of the complex were obtained by the hanging-drop vapour-diffusion technique. A complete data set to 2.5 A resolution has been collected at 100 K, using a MAR image-plate system at a synchrotron source, from crystals which belong to the space group C2 with unit-cell dimensions a = 99.38, b = 119.86, c = 77.10 A and beta = 105.56 degrees. Use of a CCD detector with cryo-cooled crystals improved the resolution to 2.3 A. A molecular replacement solution, with the 2.5 A data set, using the native SCA as a search model was obtained, with six subunits per asymmetric unit.

Agglutinins↗
PubMed1998

Crystallization and preliminary structural studies of Scilla campanulata lectin complexed with alpha1-6 mannobiose.

Recent work has shown that Scilla campanulata agglutinin from bluebell bulbs has a strong affinity for alpha(1,3)- and alpha(1,6)-linked mannosyl residues and possesses moderate antiretroviral activity. This lectin has been crystallized by the hanging-drop method of vapour diffusion complexed with the disaccharide mannose-alpha1,6-D-mannose. The crystals are in the space group P21212 with unit-cell dimensions a = 70.63, b = 92.79 and c = 47.25 A, and with a dimer in the asymmetric unit. The crystals diffract X-rays to beyond 1.5 A resolution at 277 K and are stable in an X-ray beam. Data to 1.6 A resolution have been collected using a MAR image-plate system at a synchrotron source and the structure of the complex has been solved by the molecular replacement method.

Crystallization↗
PubMed1997

Crystallization and preliminary X-ray analysis of a novel plant lectin from Calystegia sepium.

A mannose-specific lectin from Calystegia sepium has been crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The needle-shaped crystals are orthorhombic, space group C222(1) with cell dimensions a = 55.2(1), b = 55.9 (1), c = 196.1 (1) A. Fresh crystals diffract to 1.9 A resolution on a synchrotron radiation source. The asymmetric unit contains a dimer of two identical 16 kDa subunits with a packing density of 2.36 A(3) Da(-1). Intensity data have been observed beyond 2.0 A, but reasonable statistics restricted the usable range to 2.0 A.

Journal Article↗
PubMed1996

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.

Journal Article↗
PubMed1996

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.

Journal Article↗
PubMed1994

Crystallization and preliminary X-ray studies on the mannose-specific lectin from Amaryllis bulbs.

Affinity-purified amaryllis lectin was used to grow single crystals using the hanging-drop method. The space group was found to be C2 with unit-cell dimensions a = 73.4 (1), b = 100.3 (1), c = 62.2 (1) A and beta = 137.3 (2) degrees. Data to 2.25 A resolution have been recorded and solution of the structure is currently underway by means of molecular-replacement techniques.

Journal Article↗
PubMed1992

Variability in reprocessing policies and procedures for flexible fiberoptic endoscopes in Massachusetts hospitals.

BACKGROUND: Concern is increasing regarding the risk of transmission of blood-borne pathogens by means of improperly reprocessed medical devices. METHODS: On-site surveys of policies and practices were performed in 18 reprocessing areas in eight randomly selected hospitals in Massachusetts to assess current practices for reprocessing of flexible fiberoptic endoscopes. Manufacturers' and internal written protocols, were reviewed, employees were interviewed, and procedures were observed. Reprocessing included high-level disinfection in 17 of 18 areas, with 16 areas using 2% glutaraldehyde; ethylene oxide gas sterilization was used in one area. RESULTS: Considerable interhospital and intrahospital variability was found in high-level disinfection procedures, including equipment, contact time (range 10 to 45 minutes), disinfectant testing, and rinsing. Disinfection of internal channels was inadequate in three areas, recommended sterilization of biopsy forceps was not performed in five, and written protocols were unavailable in three. Ad hoc deviation from established written or verbal protocols occurred in eight areas during reprocessing of flexible fiberoptic endoscopes from patients known to have AIDS; ethylene oxide sterilization was used in seven areas and a separate device was used in one. Interviews with personnel revealed that lack of knowledge of high-level disinfection contributed to the discrepancies between policy and practice. CONCLUSIONS: We conclude that reprocessing of flexible fiberoptic endoscopes is inconsistent and potentially ineffective. Knowledge that a flexible fiberoptic endoscope was used for a patient with AIDS influences practice.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Structure of the pig insulin dimer in the cubic crystal.

Atomic coordinates for pig insulin in the cubic crystal have been refined by reciprocal-space methods to an R factor of 0.173 for data between 10.0 and 1.7 A resolution with structure-factor amplitudes greater than two standard deviations. Stereochemical parameters for the refined model are close to standard values and the estimated error in the positions of well-ordered atoms is about 0.1 A. Residues directly involved in the formation of the exact (crystallographic) cubic insulin dimer are oriented similarly to those in the non-crystallographic 2Zn insulin dimer. Other residues, which make different molecular contacts in the different crystal forms, have locally altered conformations. The cubic insulin molecule is significantly more similar to one of the two independent molecules in the 2Zn insulin dimer than the other. This more similar molecule is expected to be the more stable conformer.

Animals↗
PubMed1989

Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer.

SINCE insulin was first shown by Scott to crystallize in the presence of zinc ions in 1934, a variety of Zn-containing insulin crystals have been grown. The structures of insulin in the related rhombohedral crystals of 2Zn-insulin and 4Zn-insulin have been solved and reveal that the molecule is a hexamer, organized as three dimers, each containing a 2-fold symmetry axis and held together by Zn ions. In 2Zn-insulin the hexamer is nearly symmetrical with the two axial Zn ions and the two molecules of the dimer related closely by a local 2-fold axis. But in 4Zn-insulin the two molecules in the dimer differ remarkably, creating an asymmetric 4Zn-hexamer in which one trimer is essentially equivalent to that in 2Zn-insulin and the other is different by virtue of an additional stretch of N-terminal helix between residues B1 and B8 (refs 6, 7). We report here the structure of a new symmetrical hexamer, in which all six molecules have the B1-B8 helix seen in 4Zn-insulin. Phenol molecules, found bonding specifically to each molecule, evidently stabilize this new helical conformation.

Insulin↗
PubMed1988

The structure of 2Zn pig insulin crystals at 1.5 A resolution.

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10(-10) m = 10(-1) nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogen-bonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Structural stability in the 4-zinc human insulin hexamer.

X-ray studies on human insulins prepared by semisynthetic and biosynthetic methods have recently been undertaken. Human insulin differs from porcine insulin only at the COOH terminus of the B-chain. The present study reports the crystal structure of 4-zinc human insulin, which is used clinically as a slow-acting preparation. The structure has been refined, using 1.85-A resolution data, to a residual of 0.173. The unit cell is rhombohedral, space group R3, with hexagonal cell constants a = 80.953 and c = 37.636 A, and it is nearly isomorphous with that of 4-zinc porcine insulin. As a result of a conformational change of the first eight residues of the B-chain of molecule 1 from an extended conformation observed in the 2-zinc structure to an alpha-helical one, the coordination around one of the zinc ions on the 3-fold axis has changed, an additional zinc ion in a general position is bound by the hexamer, and additional hydrogen-bonded interactions help stabilize dimer and hexamer formation. Unlike the surface of the 2-zinc insulin hexamer, which possesses a shallow depression containing a zinc ion and its coordinating water molecules, the 4-zinc human insulin hexamer contains a zinc and chloride ion at the bottom of an 8-A tunnel produced by three parallel alpha-helices. These alpha-helices shield the zinc ion from the environment, decreasing the rate of dissociation of the hexamer, and provide an explanation for the slow-acting aspect of the 4-zinc crystalline form.

Amino Acid Sequence↗