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Biomedical subjects

A McPherson

Publications and source records attributed to A McPherson.

At least 127 records · Page 7Linked to original sources
PubMed1993

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

Amino Acid Sequence↗
PubMed1993

The three-dimensional structure of canavalin from jack bean (Canavalia ensiformis).

The three-dimensional structure of the vicilin storage protein canavalin, from Canavalia ensiformis, has been determined in a hexagonal crystal by x-ray diffraction methods. The model has been refined at 2.6 A resolution to an R factor of 0.197 with acceptable geometry. Because of proteolysis, 58 of 419 amino acids of the canavalin polypeptide are not visible in the electron density map. The canavalin subunit is composed of two extremely similar structural domains that reflect the tandem duplication observed in the cDNA and in the amino acid sequence. Each domain consists of two elements, a compact, eight-stranded beta-barrel having the "Swiss roll" topology and an extended loop containing several short alpha-helices. The root mean square deviation between 84 pairs of corresponding C alpha atoms making up the strands of the two beta-barrels in a subunit is 0.78 A, and for 112 pairs of structurally equivalent C alpha atoms of the two domains the deviation is 1.37 A. The interface between domains arises from the apposition of broad hydrophobic surfaces formed by side chains originating from one side of the beta-barrels, supplemented by at least four salt bridges. The interfaces between subunits in the trimer are supplied by the extended loop elements. These interfaces are also composed primarily of hydrophobic residues supplemented by six salt bridges. The canavalin subunits have dimensions about 40 x 40 x 86 A, and the oligomer is a disk-shaped molecule about 88 A in diameter with a thickness of about 40 A. The distribution of domains lends a high degree of pseudo-32-point group symmetry to the molecule. There is a large channel of 18 A diameter, lined predominantly by hydrophilic and charged amino acids, running through the molecule along the 3-fold axis. The majority of residues conserved between domains and among vicilins occur at the interface between subunits but appear otherwise arbitrarily distributed within the subunit, although predominantly on its exterior.

Amino Acid Sequence↗
PubMed1993

Effects of a microgravity environment on the crystallization of biological macromolecules.

Macromolecules crystals are indispensable intermediates in the analysis of macromolecular structure, are essential for the application of x-ray diffraction methods, and are at the same time the greatest obstacle to success. Protein crystals are generally difficult to grow, often of imperfect form or small size, and frequently lack sufficient order. Their growth has become the rate limiting step in x-ray crystallography. Evidence has emerged from protein crystallization experiments carried out in space that suggests macromolecular crystals of improved order and quality can be grown in a microgravity environment. Presumably the absence of density driven convection and sedimentation permits a more deliberate and graceful entry of individual molecules into the crystal lattice. This in turn results in improvements in both morphology and the diffraction patterns of the crystals. The precise mechanisms for these improvements and the means for their optimization are, however, not understood at more than a rudimentary level. I attempt here to review microgravity effects that may play a role in protein crystal growth, sedimentation, convection and surface contact, and suggest their possible mechanisms.

Animals↗
PubMed1992

The three-dimensional structure of an intact monoclonal antibody for canine lymphoma.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

Animals↗
PubMed1992

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.

Animals↗
PubMed1992

Crystallization and preliminary X-ray analysis of human endothelin.

Endothelin, a potent regulator of vasoconstriction and hypertension, is a naturally produced peptide of 21 amino acids containing two disulfide bonds. We have crystallized endothelin from humans using the vapor-diffusion technique, characterized the crystals by X-ray diffraction analysis, and have collected the X-ray intensities to a resolution of 1.8 A. The crystals, which demonstrate physical properties similar to most protein crystals and have a comparable solvent content, are hexagonal prisms that frequently grow to lengths of 400 microns and widths of 150 microns. The space group of the crystals is P6(1)22 (or P6(5)22), with a = 27.4, c = 79.6 A. There is one molecule of endothelin in the asymmetric unit of the crystals.

Crystallization↗
PubMed1992

Preliminary crystallographic study of peanut peroxidase.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.

Arachis↗
PubMed1992

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.

Crystallization↗
PubMed1991

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.

Animals↗
PubMed1991

Characterization of two crystal forms of cytochrome c from Valida membranaefaciens.

Two different crystal forms of cytochrome c from the yeast Valida membranaefaciens have been grown by vapor diffusion from polyethylene glycol 4000. Both have been characterized by X-ray diffraction. The first crystal form was grown at 277 K but proved unstable and dissolved when the temperature was increased above 288 K. This crystal was of triclinic space group P1 and had unit-cell dimensions of a = 68.4, b = 35.7, c = 33.24 A, alpha = 116.9, beta = 114.9, gamma = 75.0 degrees. Following dissolution of the triclinic crystals, the second crystal form also grew at 277 K but remained stable even at room temperature. It was of orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 27.95, b = 63.68, and c = 64.28 A. Although rather small in size, both crystal forms diffracted X-rays well and the resolution of the patterns extended beyond 3 A.

Crystallization↗
PubMed1991

Characterization of crystals of an intact monoclonal antibody for canine lymphoma.

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.

Animals↗
PubMed1991

Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens.

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 91.00 A, b = 47.17 A, c = 35.21 A and beta = 121.43 degrees. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.

Crystallization↗