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PubMed1994

Purification, crystallization and preliminary X-ray analysis of murine interleukin-5.

Wild-type and mutant forms of murine interleukin-5 (mIL-5) have been expressed in the baculovirus expression system, purified, and used in crystallization trials. Attempts to obtain diffraction quality crystals of wild-type protein were unsuccessful. The substitution of glutamine for Asn75 preserved biological activity, while removing one of two predicted N-linked glycosylation sites, and the resulting protein was crystallized from polyethylene glycol 8000 at pH 7.8 in two crystal forms. The orthorhombic crystals, which belong to space group P2(1)2(1)2 with cell dimensions a = 55.9 A, b = 83.0 A and c = 52.3 A, diffract to beyond 2.5 A resolution. The second crystal form belongs to a trigonal space group, either P3(1)21 or P3(2)21, with cell dimensions a = b = 62.1 A, c = 129.9 A, and diffracts to about 3.8 A resolution. Each crystal form probably contains one mIL-5 dimer per asymmetric unit.

Animals↗
PubMed1994

Complex between bovine ribonuclease A and porcine ribonuclease inhibitor crystallizes in a similar unit cell as free ribonuclease inhibitor.

We obtained three different morphologies of co-crystals of bovine ribonuclease A and porcine ribonuclease inhibitor. X-ray quality crystals were grown in 1.3M ammonium sulfate, 100 mM sodium acetate (pH 5.0) and 20 mM dithiothreitol at 21 degrees C. These crystals have the symmetry of the tetragonal space group I4 with a = 133.3 A and c = 86.7 A and diffract to 2.5 A resolution; they have the same symmetry and only slightly different cell parameters than the crystals of free ribonuclease inhibitor. Polyacrylamide gel electrophoresis and the crystal density indicate that both ribonuclease inhibitor and ribonuclease A are present in the crystals. Although small, crystals are suitable for three-dimensional structural analysis.

Animals↗
PubMed1995

Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure.

To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.

Base Sequence↗
PubMed1998

A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies.

When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.

Bacteriorhodopsins↗
PubMed1999

The projection structure of the membrane protein microsomal glutathione transferase at 3 A resolution as determined from two-dimensional hexagonal crystals.

The formation of two-dimensional crystals of the membrane-bound enzyme microsomal glutathione transferase is sensitive to fractional changes in the lipid-to-protein ratio. Variation of this parameter results in crystal polymorphism. The projection structure of a p6 crystal form of the enzyme has been determined by the use of electron crystallography. The unit cell at 3 A resolution is comprised of two trimers. The hexagonal p6 and the orthorhombic p21212 crystal types have common elements in the packing arrangement which imply dominant crystal contacts. An overall structural similarity between the protein molecules in the two crystal forms is suggested by the projection maps. Furthermore, a comparison of the p6 and p21212 projection maps identifies additional corresponding protein densities which could not be assigned to the microsomal glutathione transferase trimer previously. Surprisingly, an ambiguity of the rotational orientation was found for trimers interspersed at certain positions within the crystal lattice.

Animals↗
PubMed1999

Structural flexibility in human topoisomerase I revealed in multiple non-isomorphous crystal structures.

Human topoisomerase I plays a critical role in chromosomal stability by relaxing the DNA superhelical tension that arises from a variety of nuclear processes, including replication, transcription, and chromatin remodeling. Human topoisomerase I is a approximately 91 kDa enzyme composed of four major domains: a 24 kDa N-terminal domain, a56 kDa core domain, a7 kDa linker domain, and a6 kDa C-terminal domain containing the active-site Tyr723 residue. A monoclinic crystal structure of a 70 kDa N-terminally truncated form of human topoisomerase I in non-covalent complex with a 22 bp DNA duplex exhibited remarkable crystal-to-crystal non-isomorphism; shifts in cell constants of up to 9 A in the b -axis length and up to 8.5 degrees in the beta-angle were observed. Eight crystal structures of human topoisomerase I - DNA complexes from this crystal form were determined to between 2.8 and 3.25 A resolution. These structures revealed both dramatic shifts in crystal packing and functionally suggestive regions of conformational flexibility in the structure of the enzyme. Crystal packing shifts of up to 20.5 A combined with rotations of up to 11.5 degrees were observed, helping to explain the variability in cell constants. When the core subdomain III regions of the eight structures are superimposed, the "cap" (core subdomains I and II) of the molecule is observed to rotate by up to 4.6 degrees and to shift by up to 3.6 A. The linker domain shows the greatest degree of conformational flexibility, rotating and shifting by up to 2.5 degrees and 4.6 A, respectively, in five of eight structures, and becoming disordered altogether in the remaining three. These observed regions of conformational flexibility in the cap and the linker domain are consistent with the structural flexibility invoked in the "controled rotation" mechanism proposed for the relaxation of DNA superhelical tension by human topoisomerase I.

Crystallization↗
PubMed2002

X-ray diffraction of heavy-atom labelled two-dimensional crystals of rhodopsin identifies the position of cysteine 140 in helix 3 and cysteine 316 in helix 8.

We have used site-specific heavy-atom labelling and X-ray diffraction to localize single amino acid residues in the cytoplasmic domain of the integral membrane protein rhodopsin, the dim-light photoreceptor of retinal vertebrate rod cells. Two-dimensional orthorhombic crystals of the space group p22(1)2(1) (a=59.5(+/-1) A and b=82.7(+/-1.5) A) were produced from detergent-solubilized, partially delipidated rhodopsin. To obtain milligram amounts of two-dimensional crystals, which are required for X-ray diffraction, the yield of the crystalline material was significantly increased by reconstitution of rhodopsin in the presence of cholesterol (1:2 to 1:10 mol/mol) and by adding polar organic solvents to the dialysis buffer. The native cysteine residues C140 and C316 were then selectively labelled with mercury using the sulphydryl-specific reagent p-chloromercuribenzoate (1.6-2.1 mol Hg per mol rhodopsin). The labelling did not affect the unit cell dimensions. Optical absorption spectra of labelled and native two-dimensional rhodopsin crystals showed the characteristic 11-cis-retinal peak at 498 nm, which corresponds to the dark state of rhodopsin. The in-plane position of the mercury label was calculated at 9.5 A resolution from the intensity differences in the X-ray diffraction patterns of labelled and native crystals using Fourier difference methods and the phase information from electron crystallography. The label positions were in excellent agreement with the positions of C140 at the cytoplasmic end of helix 3 and of C316 in the cytoplasmic helix 8 recently obtained from three-dimensional rhodopsin crystals. Whereas these high-resolution diffraction studies were performed under cryogenic conditions (100 K), our results were obtained at room temperature with fully hydrated membranes and in the absence of loop-loop crystal contacts. To study the structural changes of the cytoplasmic loops involved in activation and signal transduction, our more physiological conditions offer important advantages. Furthermore, the localization of C316 is the first direct proof that the electron density on top of helix 1 observed by cryo-electron microscopy is a part of the C-terminal loop. Our approach is of particular interest for investigations of other membrane proteins, for which 3D crystals are not available. Structural constraints from heavy-atom labels at strategic sites enable the assignment of a position in the amino acid sequence to features visible in a low-resolution density map and the study of conformational changes associated with different functional states of the membrane protein.

Amino Acid Sequence↗
PubMedDate not supplied

Crystallization of mitochondrial creatine kinase on negatively charged lipid layers.

Polymorphic forms of crystals of mitochondrial creatine kinase (Mi-CK) octamers were generated by the lipid-layer technique, using cardiolipin as interphase adhesion matrix. Depending on the protein and lipid concentration, different types of monolayers and 3-D stacks thereof assembled in a low ionic strength crystallization buffer. Sodium tungstate was found to promote and stabilize the crystal formation, though in-plane crystallization was also possible in the absence of tungstate. All crystal forms exhibited a p4 symmetry with lattice parameters (a = b) ranging from 10.6 to 24.0 nm and with one or four octamers per unit cell in end-on orientation. In ice-embedded crystals, which showed a molecular packing different from that of negatively stained preparations, structural features of the Mi-CK octamer were observed at a resolution of 1 nm. The crystallization process took advantage of the electrostatic interaction between negatively charged lipid head groups of cardiolipin and positive charges located at the top/bottom faces of the Mi-CK octamer. In the absence of a cardiolipin support, Mi-CK formed linear filaments from a solution of phosphotungstate by association of octamers via their top/bottom faces. When tungstate was used instead of phosphotungstate, the filaments aligned themselves into large crystalline assemblies.

Animals↗
PubMed1997

Crystallization and preliminary X-ray analysis of the single-headed and double-headed motor protein kinesin.

Crystals of the single-headed and double-headed kinesin motor domains of Rattus norvegicus have been grown by vapor diffusion using ammonium sulfate as the precipitant. Both crystal systems belong to the orthorhombic space group P2(1)2(1)2(1). Double-headed kinesin crystallized with unit cell constants of a = 72.2 A, b = 91.9 A, and c = 141.7 A, and so far the best crystals diffracted to a maximum resolution of 2.7 A. Using ammonium sulfate single-headed kinesin crystallized in two different crystal forms with cell constants of a = 73.1 A, b = 73.2 A, c = 84.0 A and a = 73.4 A, b = 74.1 A, c = 74.7 A, respectively. They were found to diffract to 2.1 A resolution. Crystals of monomeric kinesin were also obtained with lithium sulfate as precipitant. They have cell constants of a = 71.6 A, b = 73.7 A, and c = 74.1 A and diffract up to 1.7 A resolution.

Animals↗
PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide.

Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.

Crystallization↗
PubMed1999

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.

Bacterial Outer Membrane Proteins↗
PubMed2000

Polymorphism in the packing of aquaporin-1 tetramers in 2-D crystals.

Hitherto, the packing arrangement of the aquaporin-1 (AQP1) tetramer in 2-dimensional (2-D) crystals (two-sided plane group p42(1)2) was observed to be largely similar (canonical crystal form) despite the difference in the source of the protein, the glycosylation state of the protein, the type of lipids, and the ratio of lipid to protein in the crystallization mixture. We report here our observation that the packing of AQP1 tetramers shows polymorphism in 2-D crystals generated in dioleoyl phosphatidylcholine bilayers. Apart from the canonical form, three additional allomorphs were identified. One was observed when small (0.25) lipid to protein ratio was used in the crystallization mixture while the other two were observed when the divalent cation content in the canonical crystals was modified. The various allomorphs were distinguished by different relative orientations of the AQP1 tetramer viewed in projection. The same, two-sided plane group p42(1)2 and similar unit cell dimensions were maintained in the different allomorphs as established by analysis of images of frozen-hydrated, nominally untilted crystals. Our results indicate that the interaction between the AQP1 monomers at the interface of the tetramers is flexible and is also strongly influenced by Mg(2+) ions with the cation effect materializing because of the intrinsic fluidity of the membrane.

Aquaporin 1↗
PubMed1991

Crystallization from concentrated sucrose solutions.

When concentrated aqueous solutions of sucrose are cooled, ice or sucrose crystals separate, depending on the concentration and temperature. The state diagram of the sucrose-water system describes both the equilibrium and non-equilibrium behaviour. A detailed account is given of the effect of sucrose concentration and various, principally polysaccharide, stabilizers on the processes of homogeneous and heterogeneous nucleation, as well as the kinetics of crystal growth. Increasing concentrations of sucrose reduce the diffusion coefficient of water molecules and the linear growth velocity of ice crystals. Polysaccharide stabilizers have relatively little effect until incipient gelation occurs. Thereafter, there is a gross change in crystal form and growth rate. A careful analysis of the impact of sucrose concentration on the rate of ice crystal growth has not permitted satisfactory matching of theoretical predictions with actual behaviour. Recent studies of the rate of growth of sucrose crystals in sucrose glasses of varying water contents are recorded. There is no simple relationship between the moisture content (in the 2-12 w/w % H2O range) and the rate of growth of the sucrose crystals.

Chemical Phenomena↗
PubMed1986

Crystal-associated matrix components in rat incisor enamel. An electron-microscopic study.

Sections of glutaraldehyde-OsO4-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcified (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.

Animals↗
PubMed1979

The effect of adenosine triphosphate, magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail, Helix pomatia L. An in vitro study.

The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl2) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO3) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0 mM ATP. The inhibitory effect of Mg2+ on CaCO3 crystal formation was diminished by reduction of the concentration of MgCl2 in the incubation solution. Thus, after incubation for only 24h, 1.0 mM MgCl2 promoted the formation of birefringent CaCO3 crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl2 and phospholipids in the recalcification of shell-repair membrane is discussed.

Adenosine Triphosphate↗
PubMed1992

Tamm-Horsfall glycoprotein--inhibitor or promoter of calcium oxalate monohydrate crystallization processes?

The processes of calcium oxalate monohydrate (COM) crystal nucleation, growth and aggregation (agglomeration) generally have been studied using a wide variety of assay systems/conditions. This paper reviews the apparently conflicting data on the effects of Tamm-Horsfall glycoprotein (THP) on COM crystallization processes in vitro, with the main emphasis on crystal aggregation. According to its well-known physico-chemical properties, THP has a dual role in modifying crystal aggregation: at high pH and low ionic strength (IS). THP is a powerful crystal aggregation inhibitor. Upon lowering pH and raising IS, THP viscosity increases, leading to reduced crystal aggregation inhibition. In the presence of additional calcium ions, some THPs even become strong promoters of crystal aggregation. This phenomenon seems to be more pronounced in THPs isolated from recurrent calcium stone formers whose proteins exhibit an abnormally high tendency of polymerization. Recent studies suggest an inherited molecular abnormality of THP among some severe recurrent calcium stone formers.

Calcium Oxalate↗
PubMed1993

The effects of citrate and urine on calcium oxalate crystal aggregation.

The rate of crystal sedimentation in a suspension of calcium oxalate monohydrate (COM) crystals was determined spectrophotometrically in the presence and absence of dialysed urine and citrate. A reduced rate of crystal sedimentation after stirring was recorded in suspensions containing citrate in concentrations between 0.33 and 1.67 mmol/l. The sedimentation rate was reduced in the presence of a 0.3-3.3% concentration of dialysed urine, with increased inhibition of crystal sedimentation when the concentration of urine was increased. A comparison of the inhibition of COM crystal sedimentation in whole urine and in dialysed urine from normal subjects and stone-formers disclosed significantly higher values (P < 0.05) in the dialysed urine. The results support previous observations that physiological concentrations of citrate might efficiently inhibit the aggregation of COM crystals. Furthermore even low concentrations of both whole urine and dialysed urine are apparently very efficient inhibitors of COM crystal aggregation.

Calcium Oxalate↗
PubMed1995

Initial enamel crystals are not spatially associated with mineralized dentine.

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adjacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.

Animals↗