Investigation of high pressure effect on the structure and adsorption of ß-lactoglobulin.

ß-Lactoglobulin, being one of the principal whey protein, is of huge importance to the food industry. Temperature/pressure effects on this small protein has been extensively studied by industry. To characterize biochemical properties of ß-lactoglobulin after or during pressurization, a wide range of methods have been used thus far. In this study, for the first time, the pressure-induced conformation of ß-lactoglobulin in the crystal state was determined, at pressure 430 MPa. Changes observed in the high pressure structure correlate with the physico-chemical properties of pressure-treated ß-lactoglobulin obtained from dynamic light scattering, electrophoretic mobility and quartz crystal microbalance with dissipation monitoring measurements. A comparison between the ß-lactoglobulin structures determined at both high and ambient pressure contrasts the stable nature of the protein core and adjacent loop fragments. At high pressure the ß-lactoglobulin structure presents early signs of dimer dissociation, charge and conformational changes characteristic for initial unfolded intermediate as well as a significant modification of the binding pocket volume. Those observations are supported by changes in zeta potential values and results in increase affinity of the ß-lactoglobulin adsorption onto gold surface. Observed pressure-induced structural modifications were previously suggested as an important factor contributing to ß-lactoglobulin denaturation process. Presented studies provide detailed analysis of pressure-associated structural changes influencing ß-lactoglobulin conformation and consequently its adsorption.

Adsorption effectiveness of ß-lactoglobulin onto gold surface determined by quartz crystal microbalance.

Bovine ß-lactoglobulin (LGB) is a transport protein that can bind to its structure hydrophobic bioactive molecules. Due to the lack of toxicity, high stability and pH-dependent molecular binding mechanism, lactoglobulin can be used as a carrier of sparingly soluble drugs. Dynamic light scattering has confirmed LGB's tendency to create oligomeric forms. The hydrodynamic diameter of LGB molecules varies from 4 nm to 6 nm in the pH range of 2-10 and ionic strength I = 0.001-0.15 M, which corresponds to the presence of mono or dimeric LGB forms. The LGB zeta potential varies from 26.5 mV to -33.3 mV for I = 0.01 M and from 13.3 mV to -16 mV for I = 0.15 M in the pH range of 2-10. The isoelectric point is at pH 4.8. As a result of strong surface charge compensation, the maximum effective ionization degree of the LGB molecule is 35% for ionic strength I = 0.01 M and 22% for I = 0.15 M. The effectiveness of adsorption is linked with the properties of the protein, as well as those of the adsorption surface. The functionalization of gold surfaces with ß-lactoglobulin (LGB) was studied using a quartz crystal microbalance with energy dissipation monitoring (QCM-D). The effectiveness of LGB adsorption correlates strongly with a charge of gold surface and the zeta potential of the molecule. The greatest value of the adsorbed mass was observed in the pH range in which LGB has a positive zeta potential values, below pH 4.8. This observation shows that electrostatic interactions play a dominant role in LGB adsorption on gold surfaces. Based on the adsorbed mass, protein orientation on gold surfaces was determined. The preferential side-on orientation of LGB molecules observed in the adsorption layer is consistent with the direction of the molecule dipole momentum determined by molecular dynamics simulations of the protein (MD). The use of the QCM-D method also allowed us to determine the effectiveness of adsorption of LGB on gold surface. Knowing the mechanism of LGB adsorption is significant importance for determining the optimum conditions for immobilizing this protein on solid surfaces. As ß-lactoglobulin is a protein that binds various ligands, the binding properties of immobilized ß-lactoglobulin can be used to design controlled protein structures for biomedical applications.

There Is High Sensitive and Specific Correlation Between Frozen and Permanent Sections in Renal Transplant Biopsies.

INTRODUCTION: Frozen sections have been used for evaluating tumors and margins during daily practice in pathology with high specificity and sensitivity (>90% for both indices both at national level and in our department). The correlation between frozen section tissue for immunofluorescent (IF) studies and permanent sections for light microscopy, along with electron microscopy, is critical for constructing a final renal pathology diagnosis. METHODS: We studied the correlation between the frozen sections for IF studies and separate fragments of tissue for permanent light microscopic sections in our renal transplant biopsies for purposes of quality control. Frozen sections for IF sections were compared with permanent sections for light microscopy in 122 renal transplant biopsies, using inflammation as the key criterion (63 with no inflammation and 59 with inflammation) to determine the correlation. RESULTS: There was high sensitivity (94.9%) and specificity (92.1%) for the correlation between the frozen section and permanent sections. CONCLUSIONS: Our data suggest that parts of renal transplant biopsy tissue dissected to freeze for IF studies and for light microscopy were highly correlated to ensure a high quality of renal tissue dissection for the final diagnosis in renal transplant biopsies.

Crystallization and preliminary crystallographic data for formyltetrahydrofolate synthetase from Clostridium thermoaceticum.

Formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been crystallized using the hanging-drop method from ammonium sulphate and PEG solutions. Crystals are trigonal, the space group is R32, a = 163 A, c = 259 A. A 4.2 A resolution data set has been collected. Analysis of the data using the self-rotation function shows that tetramers have approximate 222 symmetry and are positioned on a crystallographic 2-fold axis.

Prostatic acid phosphatase: structural aspects of inhibition by L-(+)-tartrate ions.

The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.

Flow injection amperometric determination of hydrogen peroxide by oxidation at an iridium oxide electrode.

Either an iridium electrode that was anodically pretreated or a glassy carbon electrode that was coated with a film that contained iridium oxide promoted the oxidation of hydrogen peroxide. With flow injection methodology, linear calibration curves were obtained over the 2 x 10(-5) -3.6 x 10-8M range with 0.1M KOH as the carrier. With solutions of pH 11, oxidation occurred at applied potentials of near 0.0 V vs. SCE, which is much lower than potentials needed at bare Pt or glassy carbon. The low overpotential suggests applications to systems where oxidation of other species limits the use of bare electrodes. Cyclic voltammetric studies show that HO(2)(-) is the electroactive species and that catalysis rather than mediation promotes the charge transfer. Dissolved oxygen does not interfere with the measurement.

Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu.

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism.

Crystallization and preliminary crystallographic analysis of a new crystal form of arylsulfatase A isolated from human placenta.

Depending on pH, arylsulfatase A exists in solution as a dimer or as an octamer. The enzyme isolated from human placenta was crystallized at pH 5.4 in a new crystal form with space group C2, unit-cell parameters a = 154.0, b = 190.3, c = 112.5 A, beta = 122.4 degrees and four subunits in the asymmetric unit. At pH 6.5-6.7, tetragonal crystals are obtained that are isomorphous to the known crystals of recombinant arylsulfatase A obtained at pH 5.0-5.4. The crystal structure of both forms was determined by the molecular-replacement method. The monoclinic crystals contain octamers of the same type as found in the tetragonal form.

Fluoride inhibition of yeast enolase: crystal structure of the enolase-Mg(2+)-F(-)-Pi complex at 2.6 A resolution.

Enolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase-Mg(2+)-F(-)-Pi has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/sigma (F) > or = 3 to 2.6 A resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-169, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate-enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F- and Pi most probably is due to the requirement of Pi for the closed conformation. It is possible that other Mg(2+)-dependent enzymes that have OH- ions bound to the metal ion in the transition state also will be inhibited by fluoride ions.

Model studies of the function of blockers on the small conductance potassium ion channel.

A correlation between KI (equilibrium dissociation constants) and IC50 (concentration at 50% inhibition) inhibitors for the family of blockers of the small conductance potassium ion channels and their intrinsic characteristics like molecular mass and volume have been investigated. Most of the blockers in the family are not selective, in contrast to apamin - an 18 amino acid bee venom toxin - that is known to be a highly potent and selective blocker of these channels. Differences and similarities between the blockers have been analyzed, pointing toward the origin of their selectivity and relative potency. In conclusion, an ion channel blocking is a process controlled mainly by diffusion, in accordance with previous experimental results.

Kinetic, structural and electrostatic aspects of the reduction of pentacyanoferrate(III) complexes by myoglobin.

The mechanism of the reduction of pentacyanoferrate(III) complexes by oxymyoglobin has been studied by conventional and high-pressure kinetic methods, and also by structural modelling. The results of this and an earlier study show that an outer-sphere mechanism is operating for electron transfer between oxymyoglobin and FeIII(CN)5Ln-, independent of the lability of the ligand L. The electron transfer process is preceded by precursor formation at a specific site on the protein close to the protein heme pocket.

Crystal structure of human prostatic acid phosphatase .

BACKGROUND: Prostatic acid phosphatase (hPAP) is a major product of the human prostate gland, yet its physiological substrate remains unknown. METHODS: Human PAP, purified from semen, was crystallized using polyethylene glycol as the precipitant and its crystal structure was determined using X-ray diffraction. The structure was refined at 3.1 A resolution to R = 16% and R(free) = 27%. RESULTS: The structure of hPAP is similar to that of other known histidine phosphatases, and the positions of its catalytic residues are conserved. N-linked carbohydrates are present at each of the possible glycosylation sites. It appears that high-mannose chains are attached to Asn 62 and Asp 301, while complex chains are at Asn 188. CONCLUSIONS: The similarity of the three-dimensional structures of rat PAP and human PAP indicates that the mechanistic analyses of the catalytic mechanism proposed for the rat enzyme should be extended to the human enzyme without reservations. The crystallographic data allowed the correlation of attachment sites of N-linked carbohydrate chains with a given carbohydrate type. The carbohydrates of the protein produced in the prostate cells and in the baculovirus expression system appear to differ at the site of complex carbohydrates attachment.

Ion binding induces closed conformation in Pseudomonas 7A glutaminase-asparaginase (PGA): crystal structure of the PGA-SO4(2-)-NH4+ complex at 1.7 A resolution.

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D- and L-isomers of glutamine and asparagine. X-ray quality type-1 crystals of PGA have been obtained from 2.0 M ammonium sulfate. The space group is C222(1) with unit-cell dimensions a = 78.62, b = 135.80, and c = 137.88 A. The tetrameric molecule is located on a crystallographic 2-fold axis, and two subunits form the asymmetric portion of the unit cell. The structure was solved by the molecular replacement method and refined at 1.7 A resolution to an R = 19.9% with a good geometry of the model, G = 0.05. The resultant electron density maps enabled us to resolve individual constituent atoms of most residues and introduce minor revisions to the amino acid sequence. The catalytic loop, Thr20-Gly40, is in the closed conformation with excellent electron density in both subunits. A sulfate ion and an ammonium ion are bound in the substrate binding site and interect with the loop. This interaction appears to be responsible for the observed closed conformation. New arguments supporting Thr20 as the catalytic nucleophile in the asparaginase activity are proposed.

Expression, characterization and crystallographic analysis of telluromethionyl dihydrofolate reductase.

Selenomethionine-containing proteins analyzed by multi-wavelength anomalous diffraction provide a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray crystallography [Hendrickson (1991). Science, 254, 51-58]. Since this method requires synchrotron radiation, we sought to incorporate a true heavy atom into protein, allowing the solution of the phase problem by more traditional methods of data collection. Media containing TeMet alone or TeMet with low levels of Met failed to sustain growth of a methione auxotroph of Escherichia coli carrying the dihydrofolate reductase expression vector. Growth of the organism to stationary phase and incorporation of TeMet was observed when the culture was initiated in media containing minimal Met levels and TeMet was added after induction with isopropyl-1-thio-beta-D-galactopyranoside. The purified enzyme exhibited properties similar to those of the native enzyme. Atomic absorption spectroscopy and amino-acid analysis indicated that 40% of the methionines were replaced with TeMet. Sequence analysis did not indicate significant levels of replacement in the first three sites (1, 16 and 20), suggesting that TeMet was present only in the last two sites (42 and 92). Crystals of this enzyme were grown in the presence of methotrexate and were isomorphous with crystals of wild-type dihydrofolate reductase. Difference Fourier maps and restrained least-squares refinement showed no substitution at the first three methionines, while incorporation was seen at positions 42 and 92.