Early stage mineralization in tissue engineering mapped by high resolution X-ray microdiffraction.

The specific routes of biomineralization in nature are here explored using a tissue engineering approach in which bone is formed in porous ceramic constructs seeded with bone marrow stromal cells and implanted in vivo. Unlike previous studies this model system reproduces mammalian bone formation, here investigated at high temporal resolution. Different mineralization stages were monitored at different distances from the scaffold interface so that their spatial analysis corresponded to temporal monitoring of the bone growth and mineralization processes. The micrometer spatial resolution achieved by our diffraction technique ensured highly accurate reconstruction of the different temporal mineralization steps and provided some hints to the challenging issue of the mineral deposit first formed at the organic-mineral interface. Our results indicated that in the first stage of biomineralization organic tissue provides bioavailable calcium and phosphate ions, ensuring a constant reservoir of amorphous calcium phosphate (ACP) during hydroxyapatite (HA) nanocrystal formation. In this regard we suggest a new role of ACP in HA formation, with a continuous organic-mineral transition assisted by a dynamic pool of ACP. After HA nanocrystals formed, the scaffold and collagen act as templates for nanocrystal arrangement on the microscopic and nanometric scales, respectively.

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption.

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.

The chromosomal protein sso7d of the crenarchaeon Sulfolobus solfataricus rescues aggregated proteins in an ATP hydrolysis-dependent manner.

In this work, we show that the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus displays a cation-dependent ATPase activity with a pH optimum around neutrality and a temperature optimum of 70 degrees C. Measurements of tryptophan fluorescence and experiments that used 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that ATP hydrolysis induces a conformational change in the molecule and that the binding of the nucleotide triggers the ATP hydrolysis-induced conformation of the protein to return to the native conformation. We found that Sso7d rescues previously aggregated proteins in an ATP hydrolysis-dependent manner; the native conformation of Sso7d forms a complex with the aggregates, while the ATP hydrolysis-induced conformation is incapable of this interaction. Sso7d is believed to be the first protein isolated from an archaeon capable of rescuing aggregates.

An archaeal chaperonin-based reactor for renaturation of denatured proteins.

We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock.

The probability distribution of structure factors with non-integral indices. II. The P1; case.

The probability distribution of the structure factors with non-integral indices is derived in P1;. For integral values of at least one of the indices, the intensity distribution coincides with that provided by Wilson's statistics, but may strongly differ when the indices are (or are close to) half-integers. The deviations are stronger when the integral part of the indices is small, and increase with the size of the structure. In favourable circumstances, moduli and phases of the reflections may be accurately estimated.

Annealing of complementary DNA strands above the melting point of the duplex promoted by an archaeal protein.

One enigma in the biology of hyperthermophilic microorganisms, living near or above 100 degrees C, is how their genomes can be stable and, at the same time, plastic at temperatures above the melting point. The nonspecific DNA-binding protein Sso7d of the hyperthermophilic archaeon Sulfolobus solfataricus is known to protect DNA from thermal denaturation. We report here that Sso7d promotes the renaturation of complementary DNA strands at temperatures above the melting point of the duplex. This novel annealing activity is strictly homology-dependent, and even one mismatch in a stretch of 17 complementary bases severely reduces its efficiency. Since pairing of homologous single strands is a key step in all fundamental processes involving nucleic acids, such as transcription, replication, recombination, and repair, Sso7d is a candidate component of the protein machinery devoted to the coupling of DNA stability to metabolic flexibility at high temperature.

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70 degrees C.

The biocatalysts isolated from thermophilic microorganisms are the object of ever-growing scientific interest for (i) the comprehension of the molecular basis of their thermal tolerance, and (ii) their use in different bio-industrial fields. Here we report the purification and characterization of an alcohol dehydrogenase (designated ADH-hT) from the novel strain LLD-R of Bacillus stearothermophilus which grows at 70 degrees C. ADH-hT was obtained in pure form by anion exchange chromatography and two affinity chromatographies, with a final yield of about 30%. ADH-hT was found to be a tetramer of 37 kDa-subunits, and to have a pI of 4.9. ADH-hT displayed a broad substrate specificity; its activity was highest for aldehydes, and decreased progressively for alcohols and ketones. ADH-hT was endowed with catalytic activity and resistance in the presence of several denaturing agents (organic solvents, detergents, chaotropic agents). ADH-hT shared with ADH 1503 (the alcohol dehydrogenase from B. stearothermophilus strain NCA 1503 which grows at 55 degrees C) the optimal temperature of 65 degrees C, but it was more resistant than ADH 1503 towards heating. In conclusion, due to its stability and broad substrate specificity ADH-hT could be utilized in bio-industrial processes. Furthermore, we believe that ADH-hT could represent a good model system for studying the mechanism(s) which proteins exploit to gain heat resistance.

Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule.

We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosidase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four 37.5-kDa subunits). When the proteins were heated in the presence of an equimolar amount of chaperonin, 1) the aggregation was prevented in all solutions; 2) the inactivation profiles of the single-chain enzymes were comparable with those detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperonin upon ATP hydrolysis (78 and 55% activity regains for lysozyme and alpha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin. We demonstrate by gel filtration chromatography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interaction with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis. The chaperonin had nonequivalent surfaces for the binding of the model proteins upon heating: the thermal denaturation intermediates of the single-chain proteins share Surfaces I, while the thermal denaturation intermediates of the tetrameric proteins share Surfaces II. ATP binding to the chaperonin induced a conformation that lacked Surfaces I and carried Surfaces II. These data support the concept that chaperonins protect native proteins against thermal aggregation by two mechanistically distinct strategies (an ATP-dependent strategy and an ATP-independent strategy), and provide the first evidence that a chaperonin molecule bears functionally specialized surfaces for the binding of the protein substrates.

The purification, cloning, and high level expression of a glutaredoxin-like protein from the hyperthermophilic archaeon Pyrococcus furiosus.

A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable. The first 29 amino acid residues at the N terminus of the P. furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli. The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase). The C-terminal portion of the P. furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources. The P. furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0. The expression of the P. furiosus protein, with full activity, in E. coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography. The features of the P. furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.

Isolation of a thermostable enzyme catalyzing disulfide bond formation from the archaebacterium Sulfolobus solfataricus.

A disulfide bond-forming enzyme was purified from the cytosol of the archaebacterium Sulfolobus solfataricus, strain MT-4. The enzyme, assayed by its ability to oxidize and reactivate reductively denatured ribonuclease A, had a small molecular size and displayed a high thermostability. The N-terminal amino acid sequence is reported.

Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents.

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus.

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties.

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.