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Crystallography Studies (crystallography + studies)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase

FEBS JOURNAL, Issue 2 2002
Expression, characterization, crystallization, purification
Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source]

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

PROTEIN SCIENCE, Issue 9 2004
Jennifer White
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GPI, glycophosphatidyl inositol; PpDAF, human DAF1,4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag; EcDAF, nonglycosylated human DAF 1,4 expressed in Escherichia coli; nDAF, human native glycosylated (GPI-anchored) DAF from erythrocytes; EcDAF-MP, soluble E. coli human DAF linked through a C-terminal cysteine to the myristoylated peptide APT542; PCR, polymerase chain reaction; SCR, short consensus repeat; TCEP, Tris- (2-carboxyethyl) phosphine Abstract Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli -derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. [source]

Structures of potent selective peptide mimetics bound to carboxypeptidase B

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2008
Marc Adler
This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2,nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1, pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate. [source]

A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2006
Monika Budayova-Spano
Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1,Å resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8,mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106,Å) and molecular weights (135,kDa for the homotetramer) so far successfully studied with neutrons. [source]