Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.     

Cementum protein 1‐derived peptide (CEMP 1‐p1) modulates hydroxyapatite crystal formation in vitro

A cementum protein 1‐derived peptide (CEMP1‐p1) consisting of 20 amino acids from the CEMP1's N‐terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell‐free system, was characterized. CEMP1‐p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1‐p1 were analyzed by scanning electron microscopy, Fourier‐transform infrared spectroscopy‐attenuated total reflectance (FTIR‐ATR), X‐ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1‐p1 lacks secondary structure, forms nanospheres that organize into three‐dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1‐p1 promotes the formation of spherical structures composed by densely packed prism‐like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR‐ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1‐p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.     

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk

An equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk is described in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P_i and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.Abbreviations ACP amorphous calcium phosphate - Cit citrate - CN casein - CPN calcium phosphate nanocluster - DCPD dicalcium phosphate dihydrate - HA hydroxyapatite - IAP ion activity product - MCP micellar calcium phosphate - MWCO molecular weight cut-off - OCP octacalcium phosphate - PC phosphate centre - TCC tricalcium citrate     

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.     

In Vitro Reconstitution of the Cyanobacterial Photoprotective Mechanism Mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.     

Products of estradiol/peroxidase interaction; their structural features and biopolymeric character

Loss of tritium from [2,4,6 alpha, 7 alpha-3H]estradiol and from [2-3H]estradiol during their conversion into polyestradiol (PEL) by horseradish peroxidase/H2O2 and the NMR spectrum of PEL permethyl ether suggest that PEL is composed of two or more different subunits, each formed by the joining of four molecules of estradiol with the loss of five hydrogen atoms from positions 2 and 4 and of three phenolic hydrogens leading to the formation of one C-C bond and three C-O bonds. At very low concentrations of estradiol the main reaction products were monomers; this is attributed to the initial formation of transient tetraestradiols which combine with water at high dilution and with themselves at low dilution. Association of the monomeric products to oligomers occurred on a Sephadex G-50 column and was readily reversed in phosphate buffer. In aqueous solution PEL underwent non-covalent changes induced by heat, time and electrolytes, and affecting its solubility, u.v. absorbance, extraction by organic solvents and ability to bind estradiol.     

Multiplicity of cadmium binding sites in nucleotides: X-ray evidence for the involvement of O2'' and O3'' as well as phosphate and N7 in inosine 5''-monophosphate.

Single-crystal X-ray methods have been used to characterize a hydrated polymeric cadmium derivative of inosine 5'-monophosphate. In the structure there are two independent cadmium atoms, one of which binds to two ribose oxygen atoms, an N7 position on a base, and to three water molecules. The second metal atom binds to a phosphate oxygen, three water molecules, and to two N7 atoms, which are in cis-positions. For these last; the Cd-N bonds are appreciably out of the planes of the hypoxanthine bases so that the angle between these planes is only 31.4 degrees.     

Interaction of acidic poly-amino acids with octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of poly-L-aspartate (PASP) and poly-L-glutamate (PGLU). In the absence of the polyelectrolytes, the transformation of OCP into HA is complete in 48 h. Both poly-L-aspartate and poly-L-glutamate inhibit OCP hydrolysis. However, PGLU displays a greater inhibiting effect, as a result of the different extent of phase transformation obtained at the same polyelectrolyte concentration. The inhibition takes place through polyelectrolyte adsorption on the (100) face of OCP crystals, which prevents the splitting of OCP crystals along their c-axis and the transformation into the final very long, needle-like, apatitic crystals.     

The Signaling State of Orange Carotenoid Protein

Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3′-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the β-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.     

Structure and dynamics of primary hydration shell of phosphatidylcholine bilayers at subzero temperatures.

Deuterium NMR relaxation and intensity measurements of the 2H-labeled H2O/dimyristoyl phosphatidylcholine bilayer were performed to understand the molecular origin of the freezing event of phospholipid headgroup and the structure and dynamics of unfrozen water molecules in the interbilayer space at subzero temperatures. The results suggest that about one to two water molecules associated with the phosphate group freeze during the freezing event of phospholipid headgroups, whereas about five to six waters near the trimethylammonium group behave as a water cluster and remain unfrozen at temperatures as low as -70 degrees C. In addition, temperature-dependent T1 and T2 relaxation times suggest that dynamic coupling occurs not only between the phosphate group and its bound water, but also between the methyl group and the adjacent water molecules. Based on these observations, the primary hydration shell of phosphatidylcholine headgroup at subzero temperatures is suggested to consist of two distinct regions: a clathrate-like water cluster, most likely a water pentamer, near the hydrophobic methyl group, and hydration water molecules associated with the phosphate group.     

Substituent effects on direct and indirect tautomerism of pyrimidin-2(1H)-one/pyrimidin-2-ol

The conversion of pyrimidin-2(1H)-one into pyrimidin-2-ol through direct and indirect mechanisms was investigated in the gas phase and solution media at the B3LYP/6-311++G** level of theory. The kinetic parameters demonstrate that the barrier energy ΔG^≠ of the tautomeric conversion when proton transfer is mediated by one water molecule is almost the same as when is mediated by two water molecules, and is smaller than that when is mediated by three water molecules (14.0 and 17.1?kcal/mol at 298?K, respectively). It is obvious that the indirect mechanism, which is occurred in the presence of solvent molecules, is kinetically favourable in the gas phase and aqueous media. In addition, the decrease in the ΔG^≠ values by the electron donor substituents located at the meta and para positions of pyrimidin-2(1H)-one is larger than those by the electron-withdrawing substituents.     

New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A

Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the "standard" phosphate binding site.     

The pterin (bactopterin) of carbon monoxide dehydrogenase from Pseudomonas carboxydoflava

Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. Bactopterin was three times less susceptible to air oxidation than molybdopterin. Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure.     

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.     

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.     

Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M_r 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of β-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.     

X-ray structure of the dipotassium salt of D-mannose 1-phosphate 3.25 hydrate

The first crystal structure of mannose 1-phosphate is described. The dipotassium hydrate salt crystallizes in the P2(1)2(1)2 space group. There are two independent dianions (I and II) in the asymmetric unit, which are alpha anomers adopting the 4C(1) chair conformation. The main difference between the two mannose 1-phosphate dianions is the orientation of the phosphate group with relation to the pyranosyl ring. In I, one of the phosphate oxygen atoms is antiperiplanar positions with respect to carbon atom C-1, whereas the two others are situated synclinally. The corresponding orientations of the terminal phosphate oxygen atoms in II are synperiplanar and anticlinal. The potassium cations are six- and seven-coordinate, mainly with O atoms of hydroxyl groups and water molecules. There are potassium channels extending along the c-axis. In the packing arrangement, water molecules and mannose phosphate groups also define two different types of layers parallel to a-axis. Within water channels there are extensive hydrogen-bonding networks.