Biomedical subjects

J R Luft

Publications and source records attributed to J R Luft.

25 records · Page 2Linked to original sources

PubMed1994

Time courses of equilibration for ammonium sulfate, sodium chloride and magnesium sulfate heptahydrate in the Z/3 crystallization plate.

Time courses of equilibration for three salts, sodium chloride, ammonium sulfate and magnesium sulfate heptahydrate have been measured in the Z/3 crystallization plate. It is shown that by varying both the diffusant and the reservoir depth the time taken to equilibrate can be as short as 200 or as long as 1400 h. Thus, the present design of the plate should accommodate a wide variety of desired crystallization kinetics.

Journal Article↗

PubMed1994

Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions.

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.

Amino Acid Sequence↗

PubMed1993

Structural aspects of inotropic bipyridine binding. Crystal structure determination to 1.9 A of the human serum transthyretin-milrinone complex.

The crystal structure of human transthyretin (TTR) complexed with milrinone (2-methyl-5-cyano-3,4'-bipyridin-6(1H)-one), a positive inotropic cardiac agent, has been refined to R = 17.4% for 8-1.9-A resolution data. This report provides the first detailed description of protein interactions for an inotropic bipyridine agent which is an effective thyroid hormone binding competitor to transthyretin. Milrinone is bound along the 2-fold axis in the binding site with its substituted pyridone ring located deep within the channel of the two identical binding domains of the TTR tetramer. In this orientation the 5-cyano group occupies the same site as the 3'-iodine in the TTR complex with 3,3'-diiodothyronine (Wojtczak, A., Luft, J., and Cody, V. (1992) J. Biol. Chem. 267, 353-357), which is 3.5 A deeper in the channel than thyroxine (Blake, C. C. F., and Oately, S. J., (1977) Nature 268, 115-120). These structural results confirm computer modeling studies of milrinone structural homology with thyroxine and its TTR binding interactions and explain the effectiveness of milrinone competition for thyroxine binding to TTR. To understand the weaker binding affinity of the parent inotropic drug, amrinone (5-amino-3,4'-bipyridin-6(1H)-one), modeling studies of its TTR binding were carried out which indicate that the 5-amino group cannot participate in strong interactions with TTR and the lack of the 2-methyl further weakens amrinone binding.

Binding Sites↗

PubMed1992

Crystal structure determination at 2.3-A resolution of human transthyretin-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone complex.

The crystal structure of the complex of 3',5'-dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton alpha 4 tetrameric protein of 222 molecular symmetry, as well as an inhibitor of iodothyronine deiodinase. Crystals of the TTR-bromoaurone complex are isomorphous to those of native TTR. Interpretation of difference Fourier electron density maps revealed two binding modes for the bromoaurone in each of the two independent binding sites of the TTR tetramer: deep in the channel near Ser-117 (mode I) and near the channel entrance (mode II). None of the binding modes can be fully occupied because of overlap between binding positions. A statistical disorder for bromoaurone binding was also applied, as it binds along the twofold crystallographic axis and does not possess such symmetry. The binding of mode I and that of mode II were refined at half occupancy, resulting in two molecules per tetramer. The bromoaurone binds in a nonplanar antiskewed conformation. The molecular pattern for TTR binding consists of halogen groups able to anchor between beta-sheets to form both hydrophobic and hydrophilic contacts. Comparison of structural data for bromoaurone- and thyroxine-TTR complexes indicates that bromoaurone binding mode I is 3 A deeper in the channel and binding mode II is 4 A further from the channel center than thyroxine. The bromoaurone binding observed in this TTR complex differs significantly from that based upon computer modeling studies.

Benzofurans↗

PubMed1992

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.

Escherichia coli↗

PubMed1992

Purification and crystallization of insecticidal delta-endotoxin CryIIIB2 from Bacillus thuringiensis.

CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X-ray diffraction data collected on a native crystal to 2.4 A. The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231. Crystals of the endotoxin are orthorhombic, space group C2221, with unit cell dimensions of a = 122.44, b = 131.81, and c = 105.37 A. A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit.

Bacillus thuringiensis↗

PubMed1992

Crystal structure determination at 2.3 A of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate-gamma-tetrazole.

The crystal structure of the methotrexate-gamma-tetrazole (MTXT)-NADPH ternary complex with recombinant human dihydrofolate reductase (DHFR) has been determined and refined to R = 15.9% for 7003 data from 10.0 to 2.3 A resolution for the R3 lattice. Interpretation of difference Fourier electron density maps revealed that the cofactor NADPH is bound in an extended conformation, and the closest contact between cofactor and inhibitor is 3.1 A, between N(5) of the MTXT pteridine ring and the nicotinamide C(4) which transfers a hydride during the enzyme-catalyzed reaction. As in other DHFR complexes, MTXT is interpreted as protonated at N(1) by Glu-30, and the 2-amino group is hydrogen bonded to a structurally conserved water which also interacts with Glu-30 and Thr-136. The 4-amino group of MTXT hydrogen bonds to the carbonyl of Ile-7 and the phenolic hydroxyl of Tyr-121, and the alpha-carboxylate forms a salt bridge with the conserved Arg-70. In this structure, the amide carbonyl forms two hydrogen bonds with Asn-64 and a water molecule, whereas the gamma-tetrazole ring does not interact directly with the enzyme. The largest changes in the secondary structure on formation of the ternary complex involve the fold of a flexible loop near residues 40-46, and to a lesser extent the helical region near residues 102-109 and the beta-sheet regions near residues 71-75 and 157-159.

Crystallography↗