Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Beyond the H2/CO2 upper bound: one-step crystallization and separation of nano-sized ZIF-11 by centrifugation and its application in mixed matrix membranes

The synthesis of nano-sized ZIF-11 with an average size of 36 ± 6 nm is reported. This material has been named nano-zeolitic imidazolate framework-11 (nZIF-11). It has the same chemical composition and thermal stability and analogous H2 and CO2 adsorption properties to the conventional microcrystalline ZIF-11 (i.e. 1.9 ± 0.9 μm). nZIF-11 has been obtained following the centrifugation route, typically used for solid separation, as a fast new technique (pioneering for MOFs) for obtaining nanomaterials where the temperature, time and rotation speed can easily be controlled. Compared to the traditional synthesis consisting of stirring + separation, the reaction time was lowered from several hours to a few minutes when using this centrifugation synthesis technique. Employing the same reaction time (2, 5 or 10 min), micro-sized ZIF-11 was obtained using the traditional synthesis while nano-scale ZIF-11 was achieved only by using centrifugation synthesis. The small particle size obtained for nZIF-11 allowed the use of the wet MOF sample as a colloidal suspension stable in chloroform. This helped to prepare mixed matrix membranes (MMMs) by direct addition of the membrane polymer (polyimide Matrimid®) to the colloidal suspension, avoiding particle agglomeration resulting from drying. The MMMs were tested for H2/CO2 separation, improving the pure polymer membrane performance, with permeation values of 95.9 Barrer of H2 and a H2/CO2 separation selectivity of 4.4 at 35 °C. When measured at 200 °C, these values increased to 535 Barrer and 9.1.

The ethics of the dust; ten lectures to little housewives on the elements of crystallization,

The valley of diamonds.--The pyramid builders.--The crystal life.--The crystal orders.--Crystal virtues.--Crystal quarrels.--Home virtues.--Crystal caprice.--Crystal sorrows.--The crystal rest.--Notes.

Comparison of three commercial sparse-matrix crystallization screens

Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral

Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight. These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools. An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized. Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 Angstrom resolution and belong to space group I4(1)22. The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized. These crystals belong to space group P4(2)22 and diffract to 2.4 Angstrom resolution or better.

Nanostructures, Semicrytalline Morphology, and Nanoscale Confinement Effect on the Crystallization Kinetics in Self-Organized Block Copolymer/Thermoset Blends

This work reports the first instance of self-organized thermoset blends containing diblock copolymers with a crystallizable thermoset-immiscible block. Nanostructured thermoset blends of bisphenol A-type epoxy resin (ER) and a low-molecular-weight (M-n = 1400) amphiphilic polyethylene-block-poly(ethylene oxide) (EEO) symmetric diblock copolymer were prepared using 4,4'-methylenedianiline (MDA) as curing agent and were characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). All the MDA-cured ER/EEO blends do not show macroscopic phase separation but exhibit microstructures. The ER selectively mixes with the epoxy-miscible PEO block in the EEO diblock copolymer whereas the crystallizable PE blocks that are immiscible with ER form separate microdomains at nanoscales in the blends. The PE crystals with size on nanoscales are formed and restricted within the individual spherical micelles in the nanostructured ER/EEO blends with EEO content up to 30 wt %. The spherical micelles are highly aggregated in the blends containing 40 and 50 wt % EEO. The PE dentritic crystallites exist in the blend containing 50 wt % EEO whereas the blends with even higher EEO content are completely volume-filled with PE spherulites. The semicrystalline microphase-separated lamellae in the symmetric EEO diblock copolymer are swollen in the blend with decreasing EEO content, followed by a structural transition to aggregated spherical micellar phase morphology and, eventually, spherical micellar phase morphology at the lowest EEO contents. Three morphological regimes are identified, corresponding precisely to the three regimes of crystallization kinetics of the PE blocks. The nanoscale confinement effect on the crystallization kinetics in nanostructured thermoset blends is revealed for the first time. This new phenomenon is explained on the basis of homogeneous nucleation controlled crystallization within nanoscale confined environments in the block copolymer/thermoset blends.

Phase behavior, crystallization, and morphology in thermosetting blends of a biodegradable poly(ethylene glycol)-type epoxy resin and poly(ε-caprolactone)

Thermosetting blends of a biodegradable poly(ethylene glycol)-type epoxy resin (PEG-ER) and poly(epsilon-caprolactone) (PCL) were prepared via an in situ curing reaction of poly(ethylene glycol) diglycidyl ether (PEGDGE) and maleic anhydride (MAH) in the presence of PCL. The miscibility, phase behavior, crystallization, and morphology of these blends were investigated. The uncured PCL/PEGDGE blends were miscible, mainly because of the entropic contribution, as the molecular weight of PEGDGE was very low. The crystallization and melting behavior of both PCL and the poly(ethylene glycol) (PEG) segment of PEGDGE were less affected in the uncured PCL/PEGDGE blends because of the very close glass-transition temperatures of PCL and PEGDGE. However, the cured PCL/PEG-ER blends were immiscible and exhibited two separate glass transitions, as revealed by differential scanning calorimetry and dynamic mechanical analysis. There existed two phases in the cured PCL/PEG-ER blends, that is, a PCL-rich phase and a PEG-ER crosslinked phase composed of an MAH-cured PEGDGE network. The crystallization of PCL was slightly enhanced in the cured blends because of the phase-separated nature; meanwhile, the PEG segment was highly restricted in the crosslinked network and was noncrystallizable in the cured blends. The phase structure and morphology of the cured PCL/PEG-ER blends were examined with scanning electron microscopy; a variety of phase morphologies were observed that depended on the blend composition. (C) 2004 Wiley Periodicals, Inc.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 Angstrom resolution, have unit-cell parameters a = b = 179.92, c = 185.82 Angstrom and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

Facile crystallization of Escherichia coli ketol-acid reductoisomerase

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+-dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP(+), Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 Angstrom resolution; attempts to phase them are currently in progress.

Crystallization and preliminary X-ray analysis of sulfite dehydrogenase from Stargeya novella

Crystals of purified heterodimeric sulfite dehydrogenase from Starkeya novella have been grown using vapour diffusion. X-ray diffraction data have been collected from crystals of the native protein at lambda=1.0 Angstrom and close to the iron absorption edge at lambda=1.737 Angstrom. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a=97.5, b=92.5, c=55.9 Angstrom. Native data have been recorded to 1.8 Angstrom resolution and Fe-edge data to 2.5 Angstrom.