Artificial Proteases Research Articles

Nanoproteases: Alternatives to Natural Protease for Biotechnological Applications.

Some nanomaterials with intrinsic protease-like activity have the advantages of good stability, biosafety, low price, large-scale preparation and unique property of nanomaterials, which are promising alternatives for natural proteases in various applications. An especial term, "nanoprotease", has been coined to stress the intrinsic proteolytic property of these nanomaterials. As a new generation of artificial proteases, they have become a burgeoning field, attracting many researchers to design and synthesize high performance nanoproteases. In this review, we summarize recent progress on all types of nanoproteases with regard of their activity, mechanism and application and introduce a new and effective strategy for engineering high-performance nanoproteases. In addition, we discuss the challenges and opportunities of nanoprotease research in the future.

Design and Characterization of In-One Protease-Esterase PluriZyme.

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1-15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH1AB1C, efficiently hydrolysed (azo)casein at pH 6.5-8.0 and 60-70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express.

Expanding the Scope of Polyoxometalates as Artificial Proteases towards Hydrolysis of Insoluble Proteins

Despite the enormous importance of insoluble proteins in biological processes, their structural investigation remains a challenging task. The development of artificial enzyme-like catalysts would greatly facilitate the elucidation of their structure since currently used enzymes in proteomics largely lose activity in the presence of surfactants, which are necessary to solubilize insoluble proteins. In this study, the hydrolysis of a fully insoluble protein by polyoxometalate complexes as artificial proteases in surfactant solutions is reported for the first time. The hydrolysis of zein as a model protein was investigated in the presence of Zr(IV) and Hf(IV) substituted Keggin-type polyoxometalates (POMs), (Et2 NH2 )10 [M(α-PW11 O39 )2 ] (M = Zr or Hf), and different concentrations of the anionic surfactant sodium dodecyl sulfate (SDS). Selective hydrolysis of the protein upon incubation with the catalyst was observed, and the results indicate that the hydrolytic selectivity and activity of the POM catalysts strongly depends on the concentration of surfactant. The molecular interactions between the POM catalyst and zein in the presence of SDS were explored using a combination of spectroscopic techniques which indicated competitive binding between POM and SDS towards the protein. Furthermore, the formation of micellar superstructures in ternary POM/surfactant/protein solutions has been confirmed by conductivity and Dynamic Light Scattering measurements.

Broadening the Scope of Polyoxometalates as Artificial Proteases in Surfactant Solutions: Hydrolysis of Ovalbumin by Zr(IV)-Substituted Keggin Complex

Development of catalysts for the selective hydrolysis of proteins is challenging, yet important for many applications in biotechnology and proteomics. The hydrolysis of hydrophobic proteins is particularly challenging, as due to their poor solubility, the use of surfactants is often required. In this study, the proteolytic potential of catalyst systems based on the Zr(IV)-substituted Keggin polyoxometalate (Et2NH2)10[Zr(PW11O39)2] (Zr-K 1:2) and three different surfactants (ionic SDS (sodium dodecyl sulfate); zwitterionic Zw3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)), which differ in structure and polarity, has been investigated. Hydrolysis of ovalbumin (OVA) was examined in the presence of Zr-K 1:2 and surfactants by sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), which showed the appearance of new polypeptide fragments at lower molecular weight, indicating that selective hydrolysis of OVA took place for all three catalyst systems. The same fragmentation pattern was observed, showing that the selectivity was not affected by surfactants. However, the surfactants influenced the performance of the catalyst. Hence, the interactions of OVA with surfactants and Zr-K 1:2 were investigated using different techniques such as tryptophan fluorescence, Circular Dichroism, and Dynamic Light Scattering. The speciation of the catalyst in surfactant solutions was also followed by 31P Nuclear Magnetic Resonance spectroscopy providing insight into its stability under reaction conditions.

The Dawn of Metal-Oxo Clusters as Artificial Proteases: From Discovery to the Present and Beyond.

The selective cleavage of peptide bonds in proteins is of paramount importance in many areas of the biological and medical sciences, playing a key role in protein structure/function/folding analysis, protein engineering, and targeted proteolytic drug design. Current applications that depend on selective protein hydrolysis largely rely on costly proteases such as trypsin, which are sensitive to the pH, ionic strength, and temperature conditions. Moreover, >95% of peptides deposited in databases are generated from trypsin digests, restricting the information within the analyzed proteomes. On the other hand, harsh and toxic chemical reagents such as BrCN are very active but cause permanent modifications of certain amino acid residues. Consequently, transition-metal complexes have emerged as smooth and selective artificial proteases owing to their ability to provide larger fragments and complementary structural information. In the past decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC) and pioneered a distinctive approach to the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control the regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as the protein surface charge, metal coordination to specific side chains, and hydrogen bonding between the protein surface and the MOC scaffold.In this Account, we present a critical overview of our detailed kinetic, spectroscopic, and crystallographic studies in MOC-assisted peptide bond hydrolysis, from its origins to the current rational and detailed mechanistic understanding. To this end, reactivity trends related to the structure and properties of MOCs based on the hydrolysis of small model peptides and key structural aspects governing the selectivity of protein hydrolysis are presented. Finally, our endeavors in seeking the next generation of heterogeneous MOC-based proteases are briefly discussed by embedding MOCs in metal-organic frameworks or using them as discrete nanoclusters in the development of artificial protease-like materials (i.e., nanozymes). The deep and comprehensive understanding sought experimentally and theoretically over the years in aqueous systems with intrinsic polar and charged substrates provides a unique view of the reactivity between inorganic moieties and biomolecules, thereby broadly impacting several different fields (e.g., catalysis in biochemistry, inorganic chemistry, and organic chemistry).

Expanding the reactivity of inorganic clusters towards proteins: the interplay between the redox and hydrolytic activity of Ce(iv)-substituted polyoxometalates as artificial proteases.

The ability of soluble metal-oxo clusters to specifically interact with protein surfaces makes them attractive as potential inorganic drugs and as artificial enzymes. In particular, metal-substituted polyoxometalates (MS-POMs) are remarkably selective in hydrolyzing a range of different proteins. However, the influence of MS-POMs' redox chemistry on their proteolytic activity remains virtually unexplored. Herein we report a highly site-selective hydrolysis of hemoglobin (Hb), a large tetrameric globular protein, by a Ce(iv)-substituted Keggin polyoxometalate (CeIVK), and evaluate the effect of CeIVK's redox chemistry on its reactivity and selectivity as an artificial protease. At pH 5.0, incubation of Hb with CeIVK resulted in strictly selective protein hydrolysis at six Asp-X bonds, two of which were located in the α-chain (α(Asp75-Leu76) and α(Asp94-Pro95)) and five at the β-chain (β(Asp51-Ala52), β(Asp68-Ser69), β(Asp78-Asp79), β(Asp98-Pro99) and β(Asp128-Phe129)). However, increasing the pH of the reaction mixture to 7.4 decreased the CeIVK hydrolytic reactivity towards Hb, resulting in the cleavage of only one peptide bond (β(Asp128-Phe129)). Combination of UV-Vis, circular dichroism and Trp fluorescence spectroscopy indicated similar interactions between Hb and CeIVK at both pH conditions; however, 31P NMR spectroscopy showed faster reduction of CeIVK into the hydrolytically inactive CeIIIK form in the presence of protein at pH 7.4. In agreement with these results, careful mapping of all hydrolyzed Asp-X bonds on the protein structure revealed that the lower reactivity toward the α-chain was consistent with the presence of more redox-active amino acids (Tyr and His) in this subunit in comparison with the β-chain. This points towards a link between the presence of the redox-active sites on the protein surface and efficiency and selectivity of redox-active MS-POMs as artificial proteases. More importantly, the study provides a way to tune the redox and hydrolytic reactivity of MS-POMs towards proteins through adjustment of reaction parameters like temperature and pH.

Origin of Selectivity in Protein Hydrolysis by Zr(IV)-Containing Metal Oxides as Artificial Proteases

The origin of selectivity in protein hydrolysis promoted by Zr(IV)-substituted polyoxometalates (POMs) has been investigated through a variety of computational techniques. Initially, we analyzed the reaction mechanism for the observed hydrolysis at the Asn44—Arg45 site in the hen egg-white lysozyme protein (HEWL) by the Zr-substituted Lindqvist anion [W5O18Zr(H2O)(OH)]3– (ZrL) using cluster models obtained from molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, and metadynamics simulations. The mechanism characterization shows that the overall activity is governed by the energy cost to reach the transition state for C–N bond cleavage from the reactants, resulting in a calculated overall free-energy barrier of 121 kJ mol–1 that is in excellent agreement with the values derived from the experimental rate constants (113–134 kJ mol–1). In addition, QM/MM metadynamics simulations on the early stages of the mechanism revealed the formation of an exergonic non-covalent POM···protein complex at the protein surface that was stabilized by positively charged amino acids maintained during the Zr coordination to the amide oxygen. For nonreactive related sites containing Arg (Asn113—Arg114, Arg45—Asn46, and Arg21—Gly22,) we found very similar overall barriers within the cluster model approach (124, 124, and 120 kJ mol–1, respectively); however, their nonbonding POM···protein interactions along the simulated coordination of Zr to the amide oxygen were significantly weaker than those for the reactive Asn44—Arg45 site. Thus, for the HEWL protein the selectivity is governed by an enzyme-like recognition of ZrL at the cleavage site that results in an overall acceleration of the reaction rate compared to those at other sites. Conversely for human serum albumin, (HSA) the observed selectivity was not directed by nonbonding POM···protein interactions but instead was controlled by the protein secondary structure. Calculations on several Arg—Leu sites placed in positive patches showed that peptide bonds in an α-helix structure have higher overall free-energy barriers, while for the active Arg114—Leu115 site in a random coil region the C–N cleavage is facilitated by the extended conformation of the protein chain. All in all, this study has identified and evaluated two complementary factors controlling the selectivity in peptide hydrolysis promoted by transition metal-substituted POMs; hydrolysis is disfavored at α-helical regions of the protein, and then specific positively charged patches can trap the POM via electrostatic-type POM···protein interactions and accelerate the reaction.

Hydrolysis of Peptide Bonds in Protein Micelles Promoted by a Zirconium(IV)-Substituted Polyoxometalate as an Artificial Protease.

The development of artificial proteases is challenging, but important for many applications in modern proteomics and biotechnology. The hydrolysis of hydrophobic or unstructured proteins is particularly difficult due to their poor solubility, which often requires the presence of surfactants. Herein, it is shown that a zirconium(IV)-substituted Keggin polyoxometalate (POM), (Et2 NH2 )10 [Zr(α-PW11 O39 )2 ] (1), is able to selectively hydrolyze β-casein, which is an intrinsically unstructured protein at pH 7.4 and 60 °C. Four surfactants (sodium dodecyl sulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and polyethylene glycol tert-octylphenyl ether (TX-100)), which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions, β-casein forms micellar structures in which the hydrophilic part of the protein is water accessible and able to interact with 1. Identical fragmentation patterns of β-casein in the presence of 1 were observed through SDS poly(acrylamide) gel electrophoresis both in the presence and absence of surfactants, but the rate of hydrolysis varied, depending on the nature of surfactant. Whereas TX-100 surfactant, which has a neutral polar head, caused only a slight decrease in the hydrolysis rate, stronger inhibition was observed in the presence surfactants with charges in their polar heads (CHAPS, ZW3-12, SDS). These results were consistent with those of tryptophan fluorescencequenching studies, which showed that the binding between β-casein and 1 decreased with increasing repulsion between the POM and the polar heads of the surfactants. In all cases, the micellar structure of β-casein was not significantly affected by the presence of POM or surfactants, as indicated by circular dichroism spectroscopy.

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.

Interplay between structural parameters and reactivity of Zr6-based MOFs as artificial proteases.

Structural parameters influencing the reactivity of metal–organic frameworks (MOF) are challenging to establish. However, understanding their effect is crucial to further develop their catalytic potential. Here, we uncovered a correlation between reaction kinetics and the morphological structure of MOF-nanozymes using the hydrolysis of a dipeptide under physiological pH as model reaction. Comparison of the activation parameters in the presence of NU-1000 with those observed with MOF-808 revealed the reaction outcome is largely governed by the Zr6 cluster. Additionally, its structural environment completely changes the energy profile of the hydrolysis step, resulting in a higher energy barrier ΔG‡ for NU-1000 due to a much larger ΔS‡ term. The reactivity of NU-1000 towards a hen egg white lysozyme protein under physiological pH was also evaluated, and the results pointed to a selective cleavage at only 3 peptide bonds. This showcases the potential of Zr-MOFs to be developed into heterogeneous catalysts for non-enzymatic but selective transformation of biomolecules, which are crucial for many modern applications in biotechnology and proteomics.

Total degradation of extracellular amyloids by miniature artificial proteases.

A miniaturized mimic of the active site of a protease, chymotrypsin, was linked to a target recognition unit to generate "Miniature Artificial Proteases" (mAPs). Time-resolved MALDI-TOF data analyses indicated that mAPs cleaved every amide bond between Lys16-Phe20 of the amyloid β fragment (Aβ12-21) and Aβ1-40, resulting in inhibition of fibrillization and disruption of the preformed amyloid. Such a platform may offer not only new therapeutic options against various amyloidoses but also novel routes for the selective knockdown of specific proteins.

Predominant cleavage of proteins N-terminal to serines and threonines using scandium(III) triflate

Proteolytic digestion prior to LC–MS analysis is a key step for the identification of proteins. Digestion of proteins is typically performed with trypsin, but certain proteins or important protein sequence regions might be missed using this endoproteinase. Only few alternative endoproteinases are available and chemical cleavage of proteins is rarely used. Recently, it has been reported that some metal complexes can act as artificial proteases. In particular, the Lewis acid scandium(III) triflate has been shown to catalyze the cleavage of peptide bonds to serine and threonine residues. Therefore, we investigated if this compound can also be used for the cleavage of proteins. For this purpose, several single proteins, the 20S immune-proteasome (17 proteins), and the Universal Proteomics Standard UPS1 (48 proteins) were analyzed by MALDI–MS and/or LC–MS. A high cleavage specificity N-terminal to serine and threonine residues was observed, but also additional peptides with deviating cleavage specificity were found. Scandium(III) triflate can be a useful tool in protein analysis as no other reagent has been reported yet which showed cleavage specificity within proteins to serines and threonines.Graphic abstract

Chemical Mimics of Aspartate-Directed Proteases: Predictive and Strictly Specific Hydrolysis of a Globular Protein at Asp-X Sequence Promoted by Polyoxometalate Complexes Rationalized by a Combined Experimental and Theoretical Approach.

Creating efficient and residue-directed artificial proteases is a challenging task due to the extreme inertness of the peptide bond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of a multi-subunit globular protein, hemoglobin (Hb) from bovine blood, by a range of ZrIV -substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among 570 peptide bonds in Hb, selective cleavage was observed at only eleven sites, each occurring at Asp-X peptide bonds located in the positive patches on the protein surface. The molecular origins of the observed Asp-X selectivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to ZrIV followed by a direct nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of a cyclic anhydride, which is further hydrolyzed to give the reaction products. The activation energy for the cleavage of the structurally related Glu-X sequence compared to Asp-X was calculated to be higher by 1.4 kcal mol-1 , which corresponds to a difference of about one order of magnitude in the rates of hydrolysis. The higher activation energy is attributed to the higher strain present in the six-membered ring of glutaric anhydride (Glu-X), as compared to the five-membered ring of the succinic anhydride (Asp-X) intermediate. Similarly, the cleavage at X-Asp and X-Glu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6 kcal mol-1 higher, explaining the experimentally observed selectivity. The synergy between the negatively charged polyoxometalate cluster, which binds at positive patches on protein surfaces, and selective activation of Asp-X peptide bonds located in these regions by ZrIV ions, results in a novel class of artificial proteases with aspartate-directed reactivity, which is very rare among naturally occurring proteases.

Spectroscopic Study of the Interaction between Horse Heart Myoglobin and Zirconium(IV)-Substituted Polyoxometalates as Artificial Proteases.

A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and 1 H and 31 P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 ZrIV -Keggin, exhibits the strongest interaction with HHM. 31 P NMR spectroscopy studies show that this POM dissociates in solution, resulting in the formation of a monomeric 1:1 ZrIV -Keggin structure, which is likely to be a catalytically active species. In the presence of ZrIV -POMs, HHM does not undergo complete denaturation, as evidenced by CD, UV/Vis, tryptophan fluorescence, and 1 H NMR spectroscopy. CD spectroscopy shows a gradual decrease in the α-helical content of HHM upon addition of ZrIV -POMs. The largest effect is observed in the presence of a large ZrIV -Wells-Dawson structure, whereas small ZrIV -Lindqvist POM has the least influence on the decrease in the α-helical content of HHM. In all cases, the Soret band at λ=409 nm is maintained in the presence of all examined Zr-POMs, which indicates that no conformational changes in the protein occur near the heme group.

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.

DNA-Catalyzed Amide Hydrolysis.

DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.

Interaction Study and Reactivity of Zr(IV) -Substituted Wells-Dawson Polyoxometalate towards Hydrolysis of Peptide Bonds in Surfactant Solutions.

The interaction between the 1:2 Zr(IV) :Wells-Dawson complex, K15 H[Zr(α2 -P2 W17 O61 )2] (1), and a range of surfactants was studied in detail with the aim of developing metal-substituted POMs as potential artificial proteases for membrane proteins. The surfactants include the positively charged cetyl(trimethyl)ammonium bromide (CTAB), the negatively charged sodium dodecyl sulfate (SDS), the neutral Triton X-100 (TX-100), and zwitterionic 3-[dodecyl(dimethyl)ammonio]-1-propanesulfonate (Zw3-13) and 3-[dimethyl(3-{[(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)ammonio]-1-propanesulfonate (CHAPS). A combination of multinuclear (1)H, (13)C, and (31) P NMR spectroscopy, (1)H diffusion-ordered NMR spectroscopy ((1)H DOSY), and nuclear Overhauser effect spectroscopy (NOESY) was used to examine the interaction between 1 and each surfactant on the molecular level. Cationic surfactant CTAB caused precipitation of 1 due to strong electrostatic interactions, while the anionic SDS and neutral TX-100 surfactants did not exhibit any interaction at neutral pD. (1)H DOSY NMR spectroscopy indicated an interaction between 1 and zwitterionic surfactants Zw3-12 and CHAPS, which occurs via the positively charged ammonium group in the surfactant molecule. In the presence of anionic, neutral, and zwitterionic surfactants, 1 preserves its catalytic activity towards the hydrolysis of the peptide bond in the dipeptide glycyl-l-histidine (GH). The fastest hydrolysis was observed at pD 7.0 and could be rationalized by taking into account pD-dependent speciation of 1 and coordination properties of GH.

Artificial metallopeptidases: Protein cleavage by molybdenum(VI) peroxo α-amino acid complexes

Three molybdenum(VI) peroxo α-amino acid complexes, MoO(O2)2(α-aa) (H2O) (aa=leucine, glutamine and glycine), were prepared and used as artificial proteases for site-specific cleavage of porcine pepsin. The reaction was activated by incubation of the MoO(O2)2(α-aa) (H2O)–protein mixture at 37°C (2–24h). All three molybdenum complexes resulted in different cleavage sites, indicating different binding sites on the protein. Charges and the lengths of the amino acid side chains may distribute in the variation of the probe binding sites. The study can provide a new approach for the footprinting of metal binding sites on proteins in the future.

Progress in Designing Artificial Proteases: A New Therapeutic Option for Amyloid Diseases

AbstractThis Focus Review describes the evolution of artificial proteases, which are synthetic catalysts for peptide hydrolysis that mimic the action of natural proteases. The evolution took place through discovery of catalytic centers for peptide hydrolysis, attachment of substrate‐recognition sites to the catalytic centers, and the advent of target‐selective artificial proteases. Application of the target‐selective artificial proteases led to a therapeutic option for amyloid diseases, such as Alzheimer’s disease and diabetes.

Polyoxometalates as a Novel Class of Artificial Proteases: Selective Hydrolysis of Lysozyme under Physiological pH and Temperature Promoted by a Cerium(IV) Keggin‐Type Polyoxometalate

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28-Val29 and Asn44-Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW(11)O(39))(2)](10-) polyoxometalate (POM; 1) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α-lactalbumin (α-LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the (31)P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α-helical content of the protein. (15)N-(1)H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α-helical part of the structure (Trp28-Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44-Arg45), which is found in the β-strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.