Understanding the structure-activity relationship between metal-substituted polyoxometalate complexes and biomolecules

Unraveling the three-dimensional structure and function of proteins remains of utmost importance as they serve as important drug targets. Their large size and structural complexity however complicate their structural investigations by existing experimental methods. In order to facilitate the study of proteins, most biochemical procedures require the selective hydrolysis of proteins into smaller and more manageable fragments. However, the high kinetic stability of the peptide bond, with a half-life ranging from 350 to 600 years at physiological pH, makes this a challenging procedure. This partially explains the fact that there are only a small number of commercially available selective proteases. Two main classes of commercially available proteases exist. These include proteolytical enzymes, of which trypsin is the most well known, and chemical reagents such as cyanogen bromide (CNBr). However, their various shortcomings make the search for efficient and selective artificial proteases indispensable. During the past decades, the development of metal-based artificial proteases has become an increasingly important area of research, with special focus on developing cleavage methods that are selective. To overcome the limitations of currently existing proteases, our research group recently proposed ‘‘polyoxometalates’’, also referred to as ‘‘POMs’’, as a novel class of artificial proteases. These negatively charged metal-oxygen clusters electrostatically interact with the positively charged surface patches of proteins. More importantly, incorporation of a strong Lewis acidic metal ion into the POM framework generates a metal-substituted POM, which has been proven to induce hydrolytic cleavage of proteins in a regioselective manner. This PhD thesis consists of several research projects which were conducted to get a better overall understanding on i) the biological activity of metal-substituted POMs and ii) the structure-activity relationships between metal-substituted POMs and biomolecules (proteins in particular). First of all, the factors influencing the reactivity and selectivity of a Zr-substituted Wells-Dawson POM towards an oligopeptide were explored. The specific type and order of amino acid residues together with the flexible nature of the polypeptide chain play an important role in the observed selectivity of hydrolysis. Additionally, a Zr-substituted Keggin POM was successfully cocrystallized with hen egg-white lysozyme (HEWL). The crystal structure exclusively showed the presence of a monomeric 1:1 Keggin species and three binding sites at the interface of the negatively charged POM surface and the positively charged regions of HEWL. Moreover, the hydrolytic cleavage of the globular protein, cytochrome c, was investigated by different metal-substituted POMs. It was clearly shown that the nature of the imbedded Lewis-acid metal ion played a crucial role in tuning the reactivity and selectivity during the hydrolytic process. Furthermore, it was demonstrated for the first time that a metal-substituted POM was compatible to act as a metalloprotease in the presence of a zwitterionic surfactant. Human serum albumin (HSA) was successfully hydrolyzed by a Zr-substituted Wells-Dawson POM in the presence of a zwitterionic surfactant. This result is a promising step forward towards the potential application of metal-substituted POMs as a class of artificial proteases for membrane proteins. Also, a comparison of the reactivity of the isostructural and strong Lewis acidic Zr(IV)- and Hf(IV)-substituted Wells-Dawson POMs towards the glycoprotein, ovalbumin, revealed small differences in terms of hydrolytic selectivity. Furthermore, we demonstrated that several Zr(IV)-, Hf(IV)- and Ce(IV)-based POMs are able to inhibit Taq DNA polymerase and DNA polymerase I, Klenow fragment. As DNA polymerases are important therapeutic targets, this POM-induced enzyme inhibition may be an important step forward in the search for novel therapeutic approaches.nrpages: 186status: accepte

Selective Hydrolysis of Oxidized Insulin Chain B by a Zr(IV)-Substituted Wells-Dawson Polyoxometalate

We report for the first time on the selective hydrolysis of a polypeptide system by a metal-substituted polyoxometalate (POM). Oxidized insulin chain B, a 30 amino acid polypeptide, was selectively cleaved by the Zr(IV)-substituted Wells–Dawson POM, K15H[Zr(α2-P2W17O61)2]·25H2O, under physiological pH and temperature conditions in aqueous solution. HPLC-ESI-MS, LC-MS/MS, MALDI-TOF and MALDI-TOF MS/MS data indicate hydrolysis at the Phe1–Val2, Gln4–His5, Leu6–Cys(SO3H)7, and Gly8–Ser9 peptide bonds. The rate of oxidized insulin chain B hydrolysis (0.45 h−1 at pH 7.0 and 60 °C) was calculated by fitting the integration values of its HPLC-UV signal to a first-order exponential decay function. 1H NMR measurements show significant line broadening and shifting of the polypeptide resonances upon addition of the Zr(IV)-POM, indicating that interaction between the Zr(IV)-POM and the polypeptide takes place in solution. Circular dichroism (CD) measurements clearly prove that the flexible unfolded nature of the polypeptide was retained in the presence of the Zr(IV)-POM. The thermal stability of the Zr(IV)-POM in the presence of the polypeptide chain during the hydrolytic reaction was confirmed by 31P NMR spectroscopy. Despite the highly negative charge of the Zr(IV)-POM, the mechanism of interaction appears to be dominated by a strong metal-directed binding between the positively charged Zr(IV) center and negatively charged amino acid side chains.status: publishe

Structural Characterization of the Complex between Hen Egg-White Lysozyme and Zr(IV) -Substituted Keggin Polyoxometalate as Artificial Protease

Successful co-crystallization of a noncovalent complex between hen egg-white lysozyme (HEWL) and the monomeric Zr(IV) -substituted Keggin polyoxometalate (POM) (Zr1 K1), (Et2 NH2 )3 [Zr(PW11 O39 )] (1), has been achieved, and its single-crystal X-ray structure has been determined. The dimeric Zr(IV) -substituted Keggin-type polyoxometalate (Zr1 K2), (Et2 NH2 )10 [Zr(PW11 O39 )2 ] (2), has been previously shown to exhibit remarkable selectivity towards HEWL hydrolysis. The reported X-ray structure shows that the hydrolytically active Zr(IV) -substituted Keggin POM exists as a monomeric species. Prior to hydrolysis, this monomer interacts with HEWL in the vicinity of the previously identified cleavage sites found at Trp28-Val29 and Asn44-Arg45, through water-mediated H-bonding and electrostatic interactions. Three binding sites are observed at the interface of the negatively charged Keggin POM and the positively charged regions of HEWL at: 1) Gly16, Tyr20, and Arg21; 2) Asn44, Arg45, and Asn46; and 3) Arg128.status: publishe

Structural characterization of the complex between hen egg-white lysozyme and ZrIV^{IV} -Substituted Keggin polyoxometalate as artificial protease

International audienceSuccessful co-crystallization of a noncovalent complex between hen egg-white lysozyme (HEWL) and the monomeric Zr$^{IV}$-substituted Keggin polyoxometalate(POM) (Zr1K1), (Et$_2$NH$_2$)$_3$ [Zr(PW$_{11}$O$_{39}$)] (1), has been achieved, and its single-crystal X-ray structure has been determined. The dimeric Zr$^{IV}$-substituted Keggin-type polyoxometalate (Zr1K2), (Et$_2$NH$_2$)$_{10}$[Zr(PW$_{11}$O$_{39}$)$_2$] (2), has been previously shown to exhibit remarkable selectivity towards HEWL hydrolysis. The reported X-ray structure shows that the hydrolytically active Zr$^{IV}$-substituted Keggin POM exists as a monomeric species. Prior to hydrolysis, this monomer interacts with HEWL in the vicinity of the previously identified cleavage sites found at Trp28-Val29 and Asn44-Arg45, through water-mediated H-bonding and electrostatic interactions. Three binding sites are observed at the interface of the negatively charged Keggin POM and the positively charged regions of HEWL at: 1) Gly16, Tyr20, and Arg21; 2) Asn44, Arg45, and Asn46; and 3) Arg128

Selective Hydrolysis of Ovalbumin Promoted by Hf(IV)-Substituted Wells-Dawson-Type Polyoxometalate

The reactivity and selectivity of Wells-Dawson type polyoxometalate (POM), K16[Hf(α2-P2W17O61)2]·19H2O (Hf1-WD2), have been examined with respect to the hydrolysis of ovalbumin (OVA), a storage protein consisting of 385 amino acids. The exact cleavage sites have been determined by Edman degradation experiments, which indicated that Hf1-WD2 POM selectively cleaved OVA at eight peptide bonds: Phe13-Asp14, Arg85-Asp86, Asn95-Asp96, Ala139-Asp140, Ser148-Trp149, Ala361-Asp362, Asp362-His363, and Pro364-Phe365. A combination of spectroscopic methods including 31P NMR, Circular Dichroism (CD), and Tryptophan (Trp) fluorescence spectroscopy were employed to gain better understanding of the observed selective cleavage and the underlying hydrolytic mechanism. 31P NMR spectra have shown that signals corresponding to Hf1-WD2 gradually broaden upon addition of OVA and completely disappear when the POM-protein molar ratio becomes 1:1, indicating formation of a large POM/protein complex. CD demonstrated that interactions of Hf1-WD2 with OVA in the solution do not result in protein unfolding or denaturation even upon adding an excess of POM. Trp fluorescence spectroscopy measurements revealed that the interaction of Hf1-WD2 with OVA (K q = 1.1 × 105 M-1) is both quantitatively and qualitatively slightly weaker than the interaction of isostructural Zr-containing Wells-Dawson POM (Zr1-WD2) with human serum albumin (HAS) (K q = 5.1 × 105 M-1).status: publishe

Tuning the selectivity and reactivity of metal-substituted polyoxometalates as artificial proteases by varying the nature of the embedded Lewis acid metal ion

The hydrolysis of horse heart cytochrome c (cytC) protein by two isostructural Keggin-type polyoxometalates (POMs), (Me2NH2)10[Ce(α-PW11O39)2] (Ce1–K2) and (Et2NH2)10[Zr(PW11O39)2] (Zr1–K2), which differ in the nature of the embedded Lewis acid metal ion, has been investigated. In the presence of Ce1–K2, selective hydrolysis of cytC was observed at the Trp60-Lys61 and Gly78-Thr79 peptide bonds at pH 7.4 and 37 °C. However, the isostructural Zr1–K2 exhibited a lower reactivity and different selectivity, cleaving cytC at the Asp3-Val4, Asp51-Ala52 and Gly78-Thr79 peptide bonds. Different spectroscopic techniques were used to verify the molecular interactions between cytC and each metal-substituted Keggin POM to elucidate the role of the Lewis acid metal ion in directing the selectivity of protein hydrolysis.status: publishe

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.status: publishe

Polyoxometalates as Artificial Peptidases: Reactivity towards protein targets

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