Revealing the three-dimensional murine brain microstructure by contrast-enhanced computed tomography.

To improve our understanding of the brain microstructure, high-resolution 3D imaging is used to complement classical 2D histological assessment techniques. X-ray computed tomography allows high-resolution 3D imaging, but requires methods for enhancing contrast of soft tissues. Applying contrast-enhancing staining agents (CESAs) ameliorates the X-ray attenuating properties of soft tissue constituents and is referred to as contrast-enhanced computed tomography (CECT). Despite the large number of chemical compounds that have successfully been applied as CESAs for imaging brain, they are often toxic for the researcher, destructive for the tissue and without proper characterization of affinity mechanisms. We evaluated two sets of chemically related CESAs (organic, iodinated: Hexabrix and CA4+ and inorganic polyoxometalates: 1:2 hafnium-substituted Wells-Dawson phosphotungstate and Preyssler anion), for CECT imaging of healthy murine hemispheres. We then selected the CESA (Hexabrix) that provided the highest contrast between gray and white matter and applied it to a cuprizone-induced demyelination model. Differences in the penetration rate, effect on tissue integrity and affinity for tissue constituents have been observed for the evaluated CESAs. Cuprizone-induced demyelination could be visualized and quantified after Hexabrix staining. Four new non-toxic and non-destructive CESAs to the field of brain CECT imaging were introduced. The added value of CECT was shown by successfully applying it to a cuprizone-induced demyelination model. This research will prove to be crucial for further development of CESAs for ex vivo brain CECT and 3D histopathology.

Fine-tuning non-covalent interactions between hybrid metal-oxo clusters and proteins.

Interactions between the protein Hen Egg White Lysozyme (HEWL) and three different hybrid Anderson-Evans polyoxometalate clusters - AE-NH2 (δ-[MnMo6O18{(OCH2)3CNH2}2]3-), AE-CH3 (δ-[MnMo6O18{(OCH2)3CCH3}2]3-) and AE-Biot (δ-[MnMo6O18{(OCH2)3CNHCOC9H15N2OS}2]3-) - were studied via tryptophan fluorescence spectroscopy and single crystal X-ray diffraction. Quenching of tryptophan fluorescence was observed in the presence of all three hybrid polyoxometalate clusters (HPOMs), but the extent of quenching and the binding affinity were greatly dependent on the nature of the organic groups attached to the cluster. Control experiments further revealed the synergistic effect of the anionic polyoxometalate core and organic ligands towards enhanced protein interactions. Furthermore, the protein was co-crystallised with each of the three HPOMs, resulting in four different crystal structures, thus allowing for the binding modes of HPOM-protein interactions to be investigated with near-atomic precision. All crystal structures displayed a unique mode of binding of the HPOMs to the protein, with both functionalisation and the pH of the crystallisation conditions influencing the interactions. From the crystal structures, it was determined that HPOM-protein non-covalent complexes formed through a combination of electrostatic attraction between the polyoxometalate cluster and positively charged surface regions of HEWL, and direct and water-mediated hydrogen bonds with both the metal-oxo inorganic core and the functional groups of the ligand, where possible. Hence, functionalisation of metal-oxo clusters shows great potential in tuning their interactions with proteins, which is of interest for several biomedical applications.

Hybrid assemblies of a symmetric designer protein and polyoxometalates with matching symmetry.

Novel bioinorganic hybrid materials based on proteins and inorganic clusters have enormous potential for the development of hybrid catalysts that synergistically combine properties of both materials. Here we report the creation of hybrid assemblies between a computationally designed symmetrical protein Pizza6-S and different polyoxometalates with matching symmetry: the tellurotungstic Anderson-Evans (Na6[TeW6O24]·22H2O) (TEW); Keggin (H4[SiW12O40]·6H2O) (STA); and 1 : 2 CeIII-substituted Keggin (K11[CeIII[PW11O39]2]·20H2O) (Ce-K). This results in the formation of complexes with clearly defined stoichiometries in solution. Crystal structures validate the complexes as building blocks for the formation of larger assemblies.

Redox Activity of Ce(IV)-Substituted Polyoxometalates toward Amino Acids and Peptides.

Redox reactions between polyoxometalates (POMs) and biologically relevant molecules have been virtually unexplored but are important, considering the growing interest in the biological applications of POMs. In this work we give a detailed account on the redox behavior of CeIV-substituted polyoxometalates (CeIV-POMs) toward a range of amino acids and peptides. CeIV-POMs have been shown to act as artificial proteases that promote the selective hydrolysis of peptide bonds. In presence of a protein, a concomitant reduction of CeIV to CeIII ion is frequently observed, leading us to examine the origins of this redox reaction by first using amino acid building blocks as simple models. Among all of the examined amino acids, cysteine (Cys) showed the highest activity in reducing CeIV-POMs to CeIII-POMs, followed by the aromatic amino acids tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe). While the redox reaction with Cys afforded the well-defined product cystine, no oxidation products were detected for the Trp, His, Tyr, and Phe amino acids after their reaction with CeIV-POMs, suggesting a radical pathway in which the solvent likely regenerates the amino acid. In general, the rate of redox reactions increased upon increasing the pD, temperature, and ionic strength of the reaction. Moreover, the redox reaction is highly sensitive to the type of polyoxometalate scaffold, as complexation of CeIV to a Keggin (K) or Wells-Dawson (WD) polyoxotungstate anion resulted in a large difference in the rate of redox reaction for both Cys and aromatic amino acids. The reduction of CeIVK was at least 1 order of magnitude faster in comparison to CeIVWD, in accordance with the higher redox potential of CeIVK in comparison to CeIVWD. The reaction of CeIVPOMs with a range of peptides containing redox-active amino acids revealed that the redox reaction is influenced by their coordination mode with CeIV ion, but in all examined peptides the redox reaction is favored in comparison to the hydrolytic cleavage of the peptide bond.

Direct observation of the ZrIV interaction with the carboxamide bond in a noncovalent complex between Hen Egg White Lysozyme and a Zr-substituted Keggin polyoxometalate.

The successful cocrystallization of the noncovalent complex formed between (Et2NH2)8[{α-PW11O39Zr-(µ-OH)(H2O)}2]·7H2O Keggin polyoxometalate (2) and Hen Egg White Lysozyme (HEWL) protein is reported. The resulting structural model revealed interaction between monomeric [Zr(PW11O39)]4-(1), which is a postulated catalytically active species, and the protein in two positions in the asymmetric unit. The first position (occupancy 36%) confirms the previously observed binding sites on the protein surface, whereas the second position (occupancy 14%) provides novel insights into the hydrolytic mechanisms of ZrIV-substituted polyoxometalates. The new interaction site occurs at the Asn65 residue, which is directly next to the Asp66-Gly67 peptide bond that was identified recently as a cleavage site in the polyoxometalate-catalysed hydrolysis of HEWL. Furthermore, in this newly discovered binding site, the monomeric polyoxometalate 1 is observed to bind directly to the side chain of the Asn65 residue. This binding of ZrIV as a Lewis-acid metal to the carbonyl O atom of the Asn65 side chain is very similar to the intermediate state proposed in density functional theory (DFT) studies in which ZrIV activates the peptide bond via interaction with its carbonyl O atom, and can be thus regarded as a model for interaction between ZrIV and a peptide bond.

Protein-Assisted Formation and Stabilization of Catalytically Active Polyoxometalate Species.

The effect of the protein environment on the formation and stabilization of an elusive catalytically active polyoxometalate (POM) species, K6 [Hf(α2 -P2 W17 O61 )] (1), is reported. In the co-crystal of hen egg-white lysozyme (HEWL) with 1, the catalytically active monomeric species is observed, originating from the dimeric 1:2 POM form, while it is intrinsically unstable under physiological pH conditions. The protein-assisted dissociation of the dimeric POM was rationalized by means of DFT calculations. The dissociation process is unfavorable in bulk water, but becomes favorable in the protein-POM complex due to the low dielectric response at the protein surface. The crystal structure shows that the monomeric form is stabilized by electrostatic and water-mediated hydrogen bonding interactions with the protein. It interacts at three distinct sites, close to the aspartate-containing hydrolysis sites, demonstrating high selectivity towards peptide bonds containing this residue.

Highly Selective and Tunable Protein Hydrolysis by a Polyoxometalate Complex in Surfactant Solutions: A Step toward the Development of Artificial Metalloproteases for Membrane Proteins.

This study represents the first example of protein hydrolysis at pH = 7.4 and 60 °C by a metal-substituted polyoxometalate (POM) in the presence of a zwitterionic surfactant. Edman degradation results show that in the presence of 0.5% w/v 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) detergent, a Zr(IV)-substituted Wells-Dawson-type POM, K15H[Zr(α2-P2W17O61)2]·25H2O (Zr1-WD2), selectively hydrolyzes human serum albumin exclusively at peptide bonds involving Asp or Glu residues, which contain carboxyl groups in their side chains. The selectivity and extent of protein cleavage are tuned by the CHAPS surfactant by an unfolding mechanism that provides POM access to the hydrolyzed peptide bonds.