Speciation and the structure of lead(II) in hyper-alkaline aqueous solution.

The identity of the predominating lead(ii) species in hyper-alkaline aqueous solution has been determined by Raman spectroscopy, and ab initio quantum chemical calculations and its structure has been determined by EXAFS. The observed and calculated Raman spectra for the [Pb(OH)3](-) complex are in agreement while they are different for two-coordinated complexes and complexes containing Pb[double bond, length as m-dash]O double bonds. Predicted bond lengths are also consistent with the presence of [Pb(OH)3](-) and exclude the formation of Pb[double bond, length as m-dash]O double bond(s). These observations together with experimentally established analogies between lead(ii) and tin(ii) in hyper-alkaline aqueous solutions suggest that the last stepwise hydroxido complex of lead(ii) is [Pb(OH)3](-). The Pb-O bond distance in the [Pb(OH)3](-) complex as determined is remarkably short, 2.216 Å, and has low symmetry as no multiple back-scattering is observed. The [Pb(OH)3](-) complex has most likely trigonal pyramidal geometry as all reported three-coordinated lead(ii) complexes in the solid state. From single crystal X-ray data, the bond lengths for O-coordinated lead(ii) complexes with low coordination numbers are spread over an unusually wide interval, 2.216-2.464 Å for N = 3. The Pb-O bond distance is at the short side and within the range of three coordinated complexes, as also observed for the trihydroxidostannate(ii) complex indicating that the hydroxide ion forms short bonds with d(10)s(2) metal ions with occupied anti-bonding orbitals.

Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease.

The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations.

The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7 = KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7  >> KGNK > KGNG ∼ GGNK > GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

The role of the N-terminal loop in the function of the colicin E7 nuclease domain.

Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX(14)NX(8)HX(3)H ßßα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446-576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn(2+) ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn(2+) ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485-487, 511-515 and 570-571, and in ΔN4-NColE7 those between residues 467-468, 530-535 and 570-571.

Insights into a surprising reaction: the microwave-assisted direct esterification of phosphinic acids.

It is well-known that phosphinic acids do not undergo direct esterifications with alcohols under thermal conditions. However, the esterifications take place under microwave (MW) irradiation due to the beneficial effect of MW. As a comparison, maximum 12-15% conversions were observed on traditional heating. It was proved experimentally that the MW-assisted esterifications are not reversible under the conditions applied that may be the consequence of the hydrophobic medium established by the long chain alcohol/phosphinic ester. Neither the thermodynamic, nor the kinetic data obtained by high level quantum chemical calculations justify the direct esterification of phosphinic acids under thermal conditions. The thermodynamic data show that there is no driving force for the reactions under discussion. As a consequence of the relatively high values of activation enthalpy (102-161 kJ mol(-1)), these esterifications are controlled kinetically. Comparing the energetics of the esterification of phosphinic acids and the preparative results obtained under MW conditions, one can see the potential of the MW technique in the synthesis of phosphinates. During our study, a series of new cyclic phosphinates with lipophilic alkyl groups was synthesized.

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria.

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH(2), L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH(2)L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K(D,Cu)=7.4×10(-13)M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH(2)L is coordinated by four nitrogens {NH(2),3N(im)} in the equatorial plane. In ZnH(2)L the peptide binds to zinc(II) through a {NH(2),2N(im),COO(-)} donor set, and its zinc binding affinity is relatively modest (K(D,Zn)=4.8×10(-7)M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH(2)L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH(2)L complexes has been verified by semiempirical quantum chemical calculations (PM6), too.

Theoretical design of a specific DNA-Zinc-finger protein interaction with semi-empirical quantum chemical methods.

The interactions of a zinc-finger (ZF) protein with DNA containing the specific recognition site of the ZF and with a non-specific DNA were studied with the semi-empirical quantum chemical method of PM6/Mozyme. The ZF protein (1MEY)-DNA complex structures were generated by docking calculations. The complex structures were reoptimized with the PM6/Mozyme method with implicit solvation in water. The structures were also calculated in the gas phase. The interaction enthalpies between the protein and DNA within the complexes obtained in the PM6/Mozyme with solvation optimized structures were calculated with the single-point PM6-DH2/Mozyme method (PM6 with dispersion, H-bond correction and Mozyme) with solvation. The results supported the specific and non-specific interactions in the complexes obtained from the docking experiments. The binding enthalpies of the specific and non-specific DNA binding to the protein differed significantly. The interactions between the nucleic acid strands in duplexes were also evaluated; these interactions between the base pairs were different because of the different "G…C:A…T" ratios in the DNA molecules studied. The stacking interactions between the nucleic bases were also characterized in the DNA duplexes.

Synthesis and spectroscopic and computational characterization of Zn4O(alicyclic or aromatic carboxylate)6 complexes as potential MOF precursors.

Potential metal-organic-framework precursors, Zn(4)O complexes with various alicyclic or aromatic carboxylate ligands, were prepared, in many cases quantitatively, from ZnO and the relevant carboxylic acids in the presence of trace amounts of water. The complexes obtained were characterized with various classical (titration) and instrumental (IR and NMR spectroscopies) methods and molecular modeling (PM3 and PM6 semiempirical quantum chemical methods and HF/6-31G** ab initio calculations). Structural peculiarities reflected in the success or failure in the synthesis could be rationalized with the combination of IR and NMR spectroscopies and molecular modeling.

Quantum chemical characterization of Abraham solvation parameters for gas-liquid chromatographic stationary phases.

Quantum chemical based investigation is presented on the Abraham solvation parameters for 23 molecular (non-polymeric) GLC stationary phases. PM6 semiempirical calculations combined with conductor-like screening model (COSMO) have been utilized. Comprehensive search for an optimal model was carried out, based on best subset selection from 86 variables considered. A unified quantitative structure-property relationship model has been developed for all five Abraham parameters reported. The selected set of five structure-driven descriptors was subjected to statistical analyses, and was shown to be useful for stationary phase classification.

Theoretical characterization of gas-liquid chromatographic stationary phases with quantum chemical descriptors.

Quantitative structure-property relationship (QSPR) solvent model has been developed for the McReynolds constants (prototypical solutes) on 36 gas-liquid chromatographic stationary phases. PM6 semiempirical quantum chemical calculations combined with conductor-like screening model (COSMO) has been utilized. From 276 descriptors considered, forward stepwise variable selection, followed by best subset selection, yielded linear regression models containing six purely quantum chemical and two hybrid, topologically based descriptors. Internal (leave-one-out and bootstrap) as well as external validation methods confirmed the predictive power of these structure-driven models across all 10 McReynolds constants, with 40 Kováts-index units overall root-mean-square prediction error estimate.

Penetratin and derivatives acting as antifungal agents.

The synthesis, in vitro evaluation, and conformational study of RQIKIWFQNRRMKWKK-NH(2) (penetratin) and related derivatives acting as antifungal agents are reported. Penetratin and some of its derivatives displayed antifungal activity against the human opportunistic pathogenic standardized ATCC strains Candida albicans and Cryptococcus neoformans as well as clinical isolates of C. neoformans. Among the compounds tested, penetratin along with the nonapeptide RKWRRKWKK-NH(2) and the tetrapeptide RQKK-NH(2) exhibited significant antifungal activities against the Cryptococcus species. A comprehensive conformational analysis on the peptides reported here using three different approaches, molecular mechanics, simulated annealing and molecular dynamics simulations, was carried out. The experimental and theoretical results allow us to identify a topographical template which may provide a guide for the design of new compounds with antifungal characteristics against C. neoformans.

Design of histidine containing peptides for better understanding of their coordination mode toward copper(II) by CD spectroscopy.

The systematic investigation of the copper(II) complexes of tripeptides Xaa-Xaa-His, Xaa-His-Xaa and His-Xaa-Xaa, where Xaa=Gly or Ala was performed by combined pH-metry, spectrophotometry, CD and in part EPR spectroscopy. The matrix rank analysis of the spectral data revealed the number of the coloured and optically active species as a basis for the solution speciation. A critical evaluation on the speciation and solution structure of the complexes formed is presented on the basis of their d-d band optical activity. The replacement of a Gly residue with the chiral Ala amino acid allowed us to gain decisive information on the solution structure of the complexes by CD spectroscopy. It was shown that the tripeptides with histidine in the third position formed CuH(-2)L species with (NH(2), 2N(-), ImN - where Im stands for imidazole) coordination sphere as a major species, and only the macrochelated CuL complexes as minor species around pH 5.0. In copper(II)-Xaa-His-Xaa tripeptide systems the CuH(-1)L (NH(2), N(-), ImN) is the most stable species at physiological pH, but the vacant fourth site around copper(II)ions is offered for further deprotonation, most probably resulting in mixed hydroxo species at low (<5 x 10(-4)M) metal ion concentrations, while a tetrameric complex is dominant when the copper concentration exceeds 3 x 10(-3)M. The histamine type coordination mode in CuL and CuL(2) complexes of His-Xaa-Xaa ligands predominates at low pH. The structural consequences drawn from the CD spectra for the mono and bis parent complexes were supported by theoretical calculations. CD spectra strongly suggest the participation of the imidazole nitrogen both in the Cu(2)H(-2)L(2) and CuH(-2)L complexes.

New steroid-fused P-heterocycles. Part I. Synthesis and conformational study of dioxaphosphorino[16,17-d]estrone derivatives.

D-ring-fused dioxaphosphorinanes (4-6) in the estrone series were synthetized as epimeric pairs and investigated by NMR and computational methods in order to determine their stereostructures and predominant conformations. The study was performed to evaluate the influence of the rigid sterane framework on the geometry of the condensed hetero ring, with regard to the possible steric effect of the angular methyl group at position 13. Additionally, the steric and electronic effects of the P-substituents on the conformational equilibrium were examined. The distorted-boat conformation of the hetero ring of dioxaphosphorinoestrone 3-methyl ether 4a was confirmed by single-crystal X-ray analysis. This is in good agreement with the observation in solution that, in the case of the boat conformation, the anisotropic shielding effect of the phenyl group of cyclic phosphonate 4a generates an upfield shift for 17-H, as compared with the corresponding chemical shift for epimer 4b. A similar boat conformation was substantiated for derivatives 4b, 5a, 5b and 6b on the basis of the J(H, H) and J(H, P) coupling constants and also ab initio calculations, regardless of the P-configuration. At the same time, the hetero ring of 6a seems to tilt towards a chair-like conformation due to the strong equatorial preference of the N-bis(2-chloroethyl) group.

Al(III)-binding ability of an octapeptide and its phosphorylated derivative.

A synthetic octapeptide, H-GlyGluGlyGluGlySerGlyGly-OH, and its phosphorylated Ser derivative were synthetized and their solution speciation and binding modes in their complexes with Al(III) were measured. One goal of the work was find a lead compound for the design of a selective peptide-based Al(III) chelator. pH-potentiometry was used to characterize the stoichiometry and the stability of the species formed in the interactions of the metal ion and the peptides, while multinuclear NMR was applied to characterize the binding sites of the metal ion in the complexes. CD spectroscopy revealed a difference in the conformational behaviour of the phosphorylated peptide as compared with its non-phosphorylated parent derivative. The Al(III) is presumed to enhance aggregation through the -PO3H(-)-Al(3+)-PO3(2-)-Al(3+)- intermolecular bindings between the peptide chains. The results of molecular dynamics calculations supported the experimentally obtained secondary structures and the binding position of Al(III).

Modulation of multidrug resistance and apoptosis of cancer cells by selected carotenoids.

The multidrug resistance (MDR) proteins that belong to the ATP-binding casette superfamily are present in a majority of human tumors and are an important final cause of therapeutic failure. Therefore, compounds which inhibit the function of the MDR-efflux proteins may improve the cytotoxic action of anticancer chemotherapy. The effects of carotenoids were studied on the activity of the MDR-1 gene-encoded efflux pump system. The carotenoids, isolated from paprika and other vegetables, were tested on the rhodamine 123 accumulation of human MDR-1 gene-transfected L1210 mouse lymphoma cells and human breast cancer cells MDA-MB-231 (HTB-26). Capsanthin and capsorubin enhanced the rhodamine 123 accumulation 30-fold relative to nontreated lymphoma cells. Lycopene, lutein, antheraxanthin and violaxanthin had moderate effects, while alfa- and beta-carotene had no effect on the reversal of MDR in the tumor cells. Apoptosis was induced in human MDR1 transfected mouse lymphoma cells and human breast cancer MDA-MB-231 (HTB-26) cell lines in the presence of lycopene, zeaxanthin and capsanthin. The data suggest the potential of carotenoids as possible resistance modifiers in cancer chemotherapy.

Improved mapping of protein binding sites.

Computational mapping methods place molecular probes--small molecules or functional groups--on a protein surface in order to identify the most favorable binding positions by calculating an interaction potential. Mapping is an important step in a number of flexible docking and drug design algorithms. We have developed improved algorithms for mapping protein surfaces using small organic molecules as molecular probes. The calculations reproduce the binding of eight organic solvents to lysozyme as observed by NMR, as well as the binding of four solvents to thermolysin, in good agreement with x-ray data. Application to protein tyrosine phosphatase 1B shows that the information provided by the mapping can be very useful for drug design. We also studied why the organic solvents bind in the active site of proteins, in spite of the availability of alternative pockets that can very tightly accommodate some of the probes. A possible explanation is that the binding in the relatively large active site retains a number of rotational states, and hence leads to smaller entropy loss than the binding elsewhere else. Indeed, the mapping reveals that the clusters of the ligand molecules in the protein's active site contain different rotational-translational conformers, which represent different local minima of the free energy surface. In order to study the transitions between different conformers, reaction path and molecular dynamics calculations were performed. Results show that most of the rotational states are separated by low free energy barriers at the experimental temperature, and hence the entropy of binding in the active site is expected to be high.

Identification of substrate binding sites in enzymes by computational solvent mapping.

Enzyme structures determined in organic solvents show that most organic molecules cluster in the active site, delineating the binding pocket. We have developed algorithms to perform solvent mapping computationally, rather than experimentally, by placing molecular probes (small molecules or functional groups) on a protein surface, and finding the regions with the most favorable binding free energy. The method then finds the consensus site that binds the highest number of different probes. The probe-protein interactions at this site are compared to the intermolecular interactions seen in the known complexes of the enzyme with various ligands (substrate analogs, products, and inhibitors). We have mapped thermolysin, for which experimental mapping results are also available, and six further enzymes that have no experimental mapping data, but whose binding sites are well characterized. With the exception of haloalkane dehalogenase, which binds very small substrates in a narrow channel, the consensus site found by the mapping is always a major subsite of the substrate-binding site. Furthermore, the probes at this location form hydrogen bonds and non-bonded interactions with the same residues that interact with the specific ligands of the enzyme. Thus, once the structure of an enzyme is known, computational solvent mapping can provide detailed and reliable information on its substrate-binding site. Calculations on ligand-bound and apo structures of enzymes show that the mapping results are not very sensitive to moderate variations in the protein coordinates.

Algorithms for computational solvent mapping of proteins.

Computational mapping methods place molecular probes (small molecules or functional groups) on a protein surface to identify the most favorable binding positions by calculating an interaction potential. We have developed a novel computational mapping program called CS-Map (computational solvent mapping of proteins), which differs from earlier mapping methods in three respects: (i) it initially moves the ligands on the protein surface toward regions with favorable electrostatics and desolvation, (ii) the final scoring potential accounts for desolvation, and (iii) the docked ligand positions are clustered, and the clusters are ranked on the basis of their average free energies. To understand the relative importance of these factors, we developed alternative algorithms that use the DOCK and GRAMM programs for the initial search. Because of the availability of experimental solvent mapping data, lysozyme and thermolysin are considered as test proteins. Both DOCK and GRAMM speed up the initial search, and the combined algorithms yield acceptable mapping results. However, the DOCK-based approaches place the consensus site farther from its experimentally determined position than CS-Map, primarily because of the lack of a solvation term in the initial search. The GRAMM-based program also finds the correct consensus site for thermolysin. We conclude that good sampling is the most important requirement for successful mapping, but accounting for desolvation and clustering of ligand positions also help to reduce the number of false positives.

Pentapeptide amides interfere with the aggregation of beta-amyloid peptide of Alzheimer's disease.

Amyloid peptides (Abeta) play a central role in the pathogenesis of Alzheimer's disease (AD). The aggregation of Abeta molecules leads to fibril and plaque formation. Fibrillogenesis is at the same time a marker and an indirect cause of AD. Inhibition of the aggregation of Abeta could be a realistic therapy for the illness. Beta sheet breakers (BSBs) are one type of fibrillogenesis inhibitors. The first BSB peptides were designed by Tjernberg et al. (1996) and Soto et al. (1998). These pentapeptides have proved their efficiency in vitro and in vivo. In the present study, the effects of two pentapeptide amides are reported. These compounds were designed by using the C-terminal sequence of the amyloid peptide as a template. Biological assays were applied to demonstrate efficiency. Modes of action were studied by FT-IR spectroscopy and molecular modeling methods.