Vincent Goovaerts

Molecular interactions between serum albumin proteins and Keggin type polyoxometalates studied using luminescence spectroscopy.

The interaction between the plenary Keggin H3PW12O40, lacunary Keggin K7PW11O39 and the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) type polyoxometalates (POMs), and the proteins human and bovine serum albumin (HSA and BSA) was studied using steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of the Eu-Keggin POM is dominated by a ligand-to-metal charge transfer band at 291 nm. In the absence of proteins, the number of water molecules coordinated in the first coordination sphere of the Eu(III) center of Eu-Keggin was determined to be 4, indicating that Eu(III) occurs as a 1 : 1 isomer in solution. In the presence of HSA or BSA, the number of coordinated water molecules decreased to 0 and 1, respectively, suggesting interaction between the Eu-Keggin POM and the protein surface. As a result of this interaction, a five-fold increase of the hypersensitive (5)D0 → (7)F2 transition in the luminescence intensity was observed for the Eu-Keggin-HSA complex. The association constants were calculated to be 1.5 × 10(2) M(-1) and 2.0 × 10(3) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated using a Stern-Volmer analysis. The obtained values of the quenching constants were 6.1 × 10(4) M(-1) and 2.0 × 10(6) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. The surface map of both proteins shows that the cavity containing the tryptophan has a positive surface potential, providing a specific binding site at the surface of albumin proteins for the negatively charged POM.

Molecular Origin of the Hydrolytic Activity and Fixed Regioselectivity of a ZrIV‐Substituted Polyoxotungstate as Artificial Protease

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells-Dawson polyoxometalate (POM), K15 H[Zr(α2 -P2 W17 O61 )2 ]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×10(8)  M(-1) , the three remaining sites are characterized by a lower global affinity constant of 7×10(5)  M(-1) . The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10(8)  M(-1) obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ]. In addition, Eu(III) luminescence experiments with an Eu(III) -substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu(III) due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ] have demonstrated regioselective cleavage of HSA at the Arg114Leu115, Ala257Asp258, Lys313Asp314 or Cys392Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K15 H[Zr(α2 -P2 W17 O61 )2 ].

Synthesis and Characterization of Holmium-Doped Iron Oxide Nanoparticles

Rare earth atoms exhibit several interesting properties, for example, large magnetic moments and luminescence. Introducing these atoms into a different matrix can lead to a material that shows multiple interesting effects. Holmium atoms were incorporated into an iron oxide nanoparticle and the concentration of the dopant atom was changed in order to determine its influence on the host crystal. Its magnetic and magneto-optical properties were investigated by vibrating sample magnetometry and Faraday rotation measurements. The luminescent characteristics of the material, in solution and incorporated in a polymer thin film, were probed by fluorescence experiments.

Probing Polyoxometalate–Protein Interactions Using Molecular Dynamics Simulations

The molecular interactions between the CeIV -substituted Keggin anion [PW11 O39 Ce(OH2 )4 ]3- (CeK) and hen egg-white lysozyme (HEWL) were investigated by molecular dynamics simulations. The analysis of CeK was compared with the CeIV -substituted Keggin dimer [(PW11 O39 )2 Ce]10- (CeK2 ) and the ZrIV -substituted Lindqvist anion [W5 O18 Zr(OH2 )(OH)]3- (ZrL) to understand how POM features such as shape, size, charge, or type of incorporated metal ion influence the POM⋅⋅⋅protein interactions. Simulations revealed two regions of the protein in which the CeK anion interacts strongly: cationic sites formed by Arg21 and by Arg45 and Arg68. The POMs chiefly interact with the side chains of the positively charged (arginines, lysines) and the polar uncharged residues (tyrosines, serines, aspargines) via electrostatic attraction and hydrogen bonding with the oxygen atoms of the POM framework. The CeK anion shows higher protein affinity than the CeK2 and ZrL anions, because it is less hydrophilic and it has the right size and shape for establishing interactions with several residues simultaneously. The larger, more negatively charged CeK2 anion has a high solvent-accessible surface, which is sub-optimal for the interaction, while the smaller ZrL anion is highly hydrophilic and cannot efficiently interact with several residues simultaneously.

Eu(III) luminescence and tryptophan fluorescence spectroscopy as a tool for understanding interactions between hen egg white lysozyme and metal-substituted Keggin type polyoxometalates.

The interaction between the lacunary Keggin K7PW11O39, the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) and the Ce(IV)-substituted Keggin [Me2NH2]10[Ce(PW11O39)2] (Ce-Keggin) polyoxometalates (POMs), and the proteins hen egg white lysozyme (HEWL) and the structurally homologous α-lactalbumin (α-LA) was studied by steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of Eu-Keggin at lower concentrations ([Eu-Keggin]<100 μM) is dominated by a ligand-to-metal charge transfer band (291 nm). For higher concentrations ([Eu-Keggin]>250 μM) the (5)L6←(7)F0 transition becomes the most intense peak. In the absence of protein, the number of coordinated water molecules to the Eu(III) centre of Eu-Keggin is 4, indicating a 1:1 Eu(III):POM species. In the presence of phosphate buffer this number linearly decreases from 4 to 2 upon increasing phosphate buffer concentration. Upon addition of HEWL, there are no coordinated water molecules, suggesting interaction between Eu-Keggin and the protein surface. In addition, this interaction results in a more than threefold increase of the hypersensitive (5)D0→(7)F2 transition for the Eu-Keggin/HEWL mixture. The calculated association constant amounted to 2.2×10(2) M(-1) for the Eu-Keggin/HEWL complex. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated to be 9.1×10(4) M(-1), 4×10(4) M(-1) and 4.1×10(5) M(-1) for the lacunary Keggin/HEWL, the Eu-Keggin/HEWL and the Ce-Keggin/HEWL complexes, respectively. The number of bound POM molecules to HEWL was 1.04 for the lacunary Keggin POM, and 1.0 for Eu-Keggin, indicating the formation of a 1:1 POM/HEWL complex. The value of 1.38 for Ce-Keggin might indicate a transition from 1:1 to 1:2 interaction.

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.