Analysis of solvent content and oligomeric states in protein crystals—does symmetry matter?

A nonredundant set of 9081 protein crystal structures in the Protein Data Bank was used to examine the solvent content, the number of polypeptide chains, and the oligomeric states of proteins in crystals as a function of crystal symmetry (as classified by crystal systems and space groups). It was found that there is a correlation between solvent content and crystal symmetry. Surprisingly, proteins crystallizing in lower symmetry systems have lower solvent content compared to those crystallizing in higher symmetry systems. Nevertheless, there is no universal correlation between solvent content and preferences of macromolecules to crystallize in certain space groups. Crystal symmetry as a function of oligomeric state was examined, where trimers, tetramers, and hexamers were found to prefer to crystallize in systems where the oligomer symmetry could be incorporated in the crystal symmetry. Our analysis also shows that the frequency distribution within the enantiomorphous pairs of space groups does not differ significantly, in contrast to previous reports.     

Molecular dynamics study of polyethylene chain non-isothermal crystallisation: effects of chain length and branch structure

The molecular dynamics simulations in this work were aimed to provide a molecular insight into chain structure effects on non-isothermal crystallisation of polyethylene (PE) chains. The crystallisation behaviours were influenced by chain length and cooling rate in linear PE chain crystallisation: C100 and C150 were unable to fold into crystals. From C1000 to C3000, crystallisation abilities became stronger as chain length increased. From C5000 to C14000, chain length had no influence on crystallisation abilities. Final morphologies changed from rotator phase to single crystal domain, and to multi crystal domains as chain length increased. The formation of multi crystal domains with longer chain was easier than with the shorter chain in identical conditions. Branch content influenced not only the crystallisation kinetics but also final morphologies in non-isothermal crystallisation. The branches were defective in nucleation process, which was reflected in the crystal growth process. Crystallisation temperature, rate and crystallinity decreased, and the morphologies became disordered as branch content increased. Changes of final morphologies from single crystal domain to multi crystal domains were found under the influence of branch content and cooling rate. Trans-rich phenomenon was observed, and the trans-state population increment was prior to crystallinity increment. Crystallisation processes began at different crystallisation temperature when the trans-state populations reached a critical value which was independent of branch content.     

Crystal and molecular structure of sym-homospermidine monohydrate.

Sym-homospermidine, [formula; see text] is a naturally occurring rare-polyamine found in relatively large concentration in sandal leaves. As part of our studies on structure and interactions of polyamines, sym-homospermidine was purified from sandal leaves and its structure was determined by single crystal X-ray diffraction technique. The phosphate salt of the molecule crystallized in the triclinic space group P1- with a = 8.246(1)A, b = 8.775(1)A, c = 15.531(2)A, alpha = 74.20(1) degrees, beta = 88.36(1) degrees and gamma = 65.41(1) degrees. The structure was determined by direct methods and refined to a final R factor of 5.4% for 2087 reflections with magnitude of F(obs) greater than 5 sigma [F(obs)]. The amine exists in its most favourable all trans conformation. For each amine molecule three phosphate groups exist in the crystal structure, suggesting that two of the oxygens of each phosphate group are protonated. There is also a single water molecule in the asymmetric unit in contrast to that of spermidine phosphate which has 3 water molecules. These differences probably reflect the hydrogen bonding properties of mono-ionic and di-ionic phosphate groups. The structure is predominantly stabilized by a network of hydrogen bonds.     

Crystal structure of amylose complexes with small ligands

Amylose complexes were prepared, as lamellar single crystals and polycrystalline powders, from aqueous solutions by adding small flavor molecules. The morphology, crystal structure, and thermal properties of complexes with fenchone, menthone, and geraniol were determined using transmission electron microscopy, wide-angle X-ray diffraction, and differential scanning calorimetry. The crystal structure was found to be similar to that of V amylose complexes with isopropanol. This implies that the crystallosolvates contain sixfold helices packed in orthorhombic unit cells, with the ligands possibly lying in the interhelical space. Different drying procedures were also studied leading to less resolved X-ray diagrams. The thermoanalysis confirmed that complexes with a relatively high crystallinity were formed.     

Extended form of a retro-inverso peptide stabilized by beta-sheet unidirectional H-bonds: Crystallographic and NMR evidence.

The crystallographic investigation of the retro-inverso peptide Bz-S-gAla-R-mAla-NHPh reveals an extended backbone conformation where the NH groups of the gem-diamino alkyl moiety and the CO groups of the malonyl residue face side by side. This extended conformation, presenting all carbonyls on opposite sides of the NH groups, is stabilized by interstrand H-bonds running in a single direction of the parallel beta-sheets that characterize the crystal packing. These sheets differ from the beta-sheets formed by native amino acids only. (1)H-NMR nuclear Overhauser effect spectroscopy (NOESY) experiments suggest that a conformation similar to that found in the crystal also prevails in dimethylsulfoxide solution. Previous potential energy calculations of gem-diamino alkyl (g) and malonyl (m) Ala residues predicted that extended forms were less stable than the helical ones because of strong electrostatic repulsions between the parallel polar groups. Similar arguments were invoked to give more weight to helical forms of the retro-peptide units in the proposal of packing models of some nylons in their crystalline polar regions. The present findings show that both g and m Ala residues can experience the extended conformation in the beta-sheet aggregation. The energy increase occurring in one strand, due to the parallel orientation of consecutive peptide dipoles, is more than compensated by favorable cooperative interactions among head-to-tail aligned peptide dipoles of facing strands, resulting in the formation of two C==O...H==N H-bonds per residue.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to transition metal ions

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with various transition metal ions were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. Two CuCl2-erythritol complexes (denoted as CuE(I) and CuE(II)) were obtained. In CuE(I), Cu2+ coordinates with two chloride ions and four OH groups from two erythritol molecules. Two copper centers are linked by one erythritol molecule to form a zigzag chain. For CuE(II), each Cu2+ coordinates with two OH groups from an erythritol molecule and two chloride ions. The crystal of CuE(II) contains complexed and free erythritol, the dimers of [Cu2Cl4(C4H10O4)] further form a [Cu2Cl4(C4H10O4)]infinity chain via secondary Cu...Cl bonds, both the dimer unit of [Cu2Cl4.(C4H10O4)] and non-coordinated C4H10O4 unit exist side by side in the crystal. MnCl2-erythritol complex whose structure is similar to CuE(I) is also acquired. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network that link chain and layer together to build three-dimensional structures.     

Pyrazolopyridine inhibitors of B-Raf(V600E). Part 3: an increase in aqueous solubility via the disruption of crystal packing

A single crystal was obtained of a lead B-Raf(V600E) inhibitor with low aqueous solubility. The X-ray crystal structure revealed hydrogen-bonded head-to-tail dimers formed by the pyrazolopyridine and sulfonamide groups of a pair of molecules. This observation suggested a medicinal chemistry strategy to disrupt crystal packing and reduce the high crystal lattice energy of alternative inhibitors. Both a bulkier group at the interface of the dimer and an out-of-plane substituent were required to decrease the compound's melting point and increase aqueous solubility. These substituents were selected based on previously developed structure-activity relationships so as to concurrently maintain good enzymatic and cellular activity against B-Raf(V600E).     

Nuclear magnetic resonance of heme protein crystals. General aspects

A new technique capable of determining the static and dynamic structures of heme protein crystals is reported. It is shown that microcrystals of a variety of paramagnetic heme proteins, suspended in approximately 90% saturated (NH4)2SO4, may be perfectly aligned by an intense static external magnetic field, H0, due to the large anisotropy in the magnetic susceptibility of the protein caused by the paramagnetic center. Myoglobin from sperm whale (Physeter catodon) was isotopically enriched at the C epsilon methyl groups of methionine residues 55 and 131 with either 13C or 2H and studied in the crystalline solid state by 2H-quadrupole echo and 13C-Fourier transform nuclear magnetic resonance spectroscopy. It was found that suspensions of both high (S = 5/2) and low (S = 1/2) spin ferric forms of the labeled protein were ordered, the axis of ordering being approximately perpendicular to the low temperature minimum g tensor valve, even though upper Kramers levels are populated at room temperature. The paramagnetic CoII derivative "coboglobin" showed similar ordering behavior, but the diamagnetic carboxymyoglobin was unaffected. The magnetic ordering method permits the recording of "single crystal" NMR spectra from microcrystalline arrays of proteins which cannot be prepared in large enough form (approximately 1 cm3) for single crystal NMR spectroscopy and thereby allows the resolution and assignment of numerous single atom sites in the crystalline solid state. The information from a "single crystal" NMR spectrum combined with that obtained on the crystal powder allows for the direct determination of (i) the spatial orientation of the particular labeled residue within the protein crystal and (ii) the rates and types of side chain motion. Resonances were assigned by spin label broadening experiments and by use of existing x-ray data to predict 2H-NMR spectra. This new technique opens up the possibility of determining directly the dynamic structure of protein crystals and of comparing the structures of proteins in the crystalline solid state with that in solution and is applicable to other heme proteins, e.g. catalase.     

The physical action of cellulases revealed by a quartz crystal microbalance study using ultrathin cellulose films and pure cellulases

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.     

An unusual electron trapping site in the crystal structure of dulcitol

Electrons are trapped in intermolecular voids in single crystals of dulcitol X-irradiated at low temperature. The hyperfine interactions between the trapped electron and the protons of the hydroxy groups which form the trap suggest a highly symmetrical arrangement of two hydroxy groups in apposition. On the basis of this consideration the site in the crystal structure where trapping occurs was identified. In a parallel approach the crystal structure of dulcitol was surveyed for a suitable void encompassing a positive potential. The latter approach confirmed the same site in the crystal structure as the electron trapping site.     

Studies of electrons trapped in X-irradiated rhamnose crystals

The electrons trapped in single crystals of rhamnose X-irradiated at low temperature were studied by ENDOR spectroscopy. Hyperfine couplings of protons in the environs of the electron have been determined from ENDOR measurements, including those of some of the more remote carbon-bound hydrogen atoms. The likely site of electron trapping in the crystal structure of rhamnose was inferred from calculations of the electric potential generated by the dipoles of hydroxy groups about preexisting void spaces. Electron-proton distances for nonexchangeable hydrogen atoms from points within the void were calculated from the crystal structure and compared with distances obtained from hyperfine couplings. Good agreement was obtained between experimental and calculated values.     

Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules

We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10(-3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.     

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone at 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.     

Catalysis of a rotational transition in a peptide by crystal forces

Detailed examination of the dynamics trajectories of the isolated cyclic peptide cyclo-(Ala-Pro-D-Phe)2 and of the molecule in its crystalline environment led to the unexpected observation that the methyl groups of the alanine residues rotated more frequently during a simulation in the crystal environment than in a simulation of the isolated peptide. In effect, the crystal environment is "catalyzing" the rotational isomerization of the methyl groups. In order to understand how the crystal forces increase the rate of this rotation, and to explore any possible analogy to the inducing of strained conformations of ligands by enzymes, the barriers to rotation in the two environments were studied by using the torsion angle forcing method. The crystal forces induce a different, higher energy, conformation of the peptide than is found for the isolated molecule, and the different rates of rotation have been explained in terms of the resulting specific intramolecular interactions that, it turns out, give rise to the lower rotational barrier. Molecular dynamics simulations of the peptide were also run at higher temperatures in order to calculate the barriers to rotation through the use of Arrhenius plots. The barriers obtained in this way agree well with the barriers obtained through an adiabatic reaction path derived by rotating the methyl through the barrier while minimizing all remaining degrees of freedom. The rates of rotation calculated from these adiabatic barriers also agree well with the rates observed during the 300 K simulations.     

Conformational,configurational, and supramolecular studies of podocephalol acetate from Lasianthaea aurea

Although podocephalol ( 1 ) and its derived acetate 2 were found in Lasianthaea podocephala four decades ago, and 1 was later detected in the essential oils of several vegetal species, its absolute configuration (AC) and conformational preferences remained to be established. The structures of ar‐himachalene 1 , now isolated from Lasianthaea aurea, and its derived acetate 2 , were herein confirmed by extensive 1D and 2D nuclear magnetic resonance (NMR) studies, while the conformational preferences of the cycloheptene was established by density functional theory (DFT) calculations, which in combination with vibrational circular dichroism measurements provided the (R) absolute configuration of the molecules. The structure and AC were further verified through the Flack and Hooft parameters calculations derived from single crystal X‐ray diffraction data of 2 . In addition, careful evaluation of the crystal data allowed observing supramolecular layers cell package, an uncommon property in natural terpenes that might have potential applications. A transmission electron microscopy analysis of crystal of 2 was also possible, providing its physical characteristics at the micrometric scale.     

NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.

Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.     

Structure, interaction, dynamics and solvent effects on the DNA-EcoRI complex in aqueous solution from molecular dynamics simulation.

A 0.7-ns molecular dynamics simulation of the DNA-EcoRI complex in a 7.0-A solvent shell indicated a stable behavior of the system. No significant evaporation or smearing of the solvent's outer boundary occurred. The structure and the intermolecular interactions were found to be well maintained during the simulation. The interaction pattern in the simulation was found to be very similar to that in the crystal structure. Most of the specific interactions between the DNA and the protein were found to be enhanced in the simulation compared to that in the crystal structure as a result of improved interaction geometry. The nonspecific interactions were found to be stronger than the specific ones. The specific interactions between the N7 atoms of Gua(4) or Ade(5) or Ade(6) and the protein were found to be present over almost the entire time of the simulation, whereas hydrogen bonds involving the amino groups of the Ade(5) and Ade(6) with the protein were found to be relatively weaker, with lower probability and shorter lifetime. The time evolution of the root mean square deviations of the DNA and the protein were highly correlated even at the later part of the simulation, showing the tight binding between them. Several long-lived water bridges were found between the DNA backbone atoms and the protein and also between the two protein monomers, which increased the overall stability of the complex. The two protein monomers were found to interact strongly with each other. The energy of the DNA kink deformation was estimated as approximately 31 kcal/mol.     

Crystal structure and conformation of a cyclic tetrapeptide cyclo(L-Pro-Sar)2 containing all-cis peptide units

The crystal structure and conformation of the synthetic cyclic tetrapeptide, cyclo(L -Pro-Sar)₂, was determined by x-ray analysis. The peptide crystallizes in the orthorhombic space group P2₁2₁2₁ with cell parameters a = 9.277(1), b = 12.884(1), and c = 15.581(2) Å. The crystal structure was solved by the symbolic addition procedure for direct phase determination and least-squares refinement using 1796 reflections, which led to the final R value of 0.043. This structure provides the first example observed in a crystal of a cyclic tetrapeptide in which all four peptide units have been found in the cis conformation with ω angles deviating slightly by 2°–10° from the ideal value of 0°. It was also found that the two Pro C^α-CO single bonds assumed a trans′ (ψ = 159.6° and 158.4°) conformation. Adjoining average planes of the peptide groups fall at nearly right angles to each other. The pyrrolidine ring conformations of the two prolyl residues are in the envelope form, with C^γ carbon out of the least-squares planes for the remaining four atoms.     

虎掌（天南星科）中草酸钙晶体的形态和分布

在光学显微镜下对虎掌（Pinellia pedatisecta）营养器官和繁殖器官中晶体的类型和分布进行了观察和分析,探讨晶体的功能与作用机制。结果表明：（1）虎掌各个器官中都发现有晶体,且晶体类型有针晶、簇晶、砂晶和柱晶4种形态,其中针晶最为常见。（2）虎掌叶中的晶体大多以针晶状分布在叶片上表皮下的叶肉中,少数分布在叶下表皮下的叶肉中,其次砂晶和星芒状簇晶也在叶中较常见,叶中也有少量的柱晶。（3）虎掌的块茎中分布有大量的针晶束,在输导组织附近还有一些大的簇晶;虎掌的不定根中分布有不整齐的针晶和排列不规则的针晶束以及少量大的簇晶。（4）虎掌的佛焰苞中分布有针晶、簇晶和砂晶,且在佛焰苞中的针晶主要分布于上、下表皮之下的叶肉中,砂晶多分布在佛焰苞的上、下表皮上。（5）虎掌的花药壁中分布有针晶束,其方向和花药壁表面垂直,而花粉囊中只有小的簇晶。（6）虎掌的果皮和种皮上分布有大量的针晶。根据晶体在酸中的溶解性,虎掌体内所有晶体的化学成分都为草酸钙。研究认为,虎掌各个器官中的各种草酸钙晶体对于保护虎掌免受食草动物取食具有重要的作用。     

Diversity of locations for Bacillus thuringiensis crystal protein genes.

The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.