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

Abstract

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), (Et2NH2)10[Zr(α‐PW11O39)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.

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

“…Upon addition of increasing amounts of Zr‐K 1 : 2 to zein in the presence of SDS, the negative peak at 208 nm decreased towards zero and eventually disappeared while the peak at 222 nm only decreased slightly (Figure 5; Figure S8). This decrease in the peaks and change in the CD 222 /CD 208 ratio suggests a restructuring of the protein structure which may be due to changes in the secondary and tertiary structure of zein induced by the POM as has been previously reported for other proteins [23,24] . Unfolding of the protein may also be influenced by repulsive interactions between SDS and the negatively charged POM when both are in the vicinity of the protein surface.…”

“…The hydrolysis of zein, as a model insoluble protein, was investigated in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), one of the most commonly used surfactants for the solubilization of membrane proteins (Figure 1 (a)) [9] . Zr(IV) and Hf(IV) substituted Keggin‐type POMs ((Et 2 NH 2 ) 10 [M(α‐PW 11 O 39 ) 2 ] where M = Zr or Hf; M‐K 1 : 2) were used to promote the hydrolysis reaction since they have been previously shown to be active in the hydrolysis of several water‐soluble proteins (Figure 1 (b)) [15,22–24] …”

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

“…The hydrolysis reaction typically proceeds under mild pH conditions and produces large peptide fragments (3-15 kDa) that are well suited for applications in middle-down proteomic studies [5][6][7][8][9][10][11][12][13][14]. Furthermore, MS-POMs have been shown to maintain their activity towards peptide bond hydrolysis in surfactant solutions [18][19][20][21]. Interestingly, these studies with model proteins have shown that the extent to which surfactants influence the reactivity and selectivity of protein hydrolysis is largely dependent on the type of protein.…”

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

“…[15] Furthermore, it was found that the catalytic activity of metal-substituted POMs is preserved in surfactant solutions, where the hydrolysis of peptides and different watersoluble globular proteins, [12,[21][22][23] as well as of a partially soluble unstructured protein has been observed. [24] These studies revealed that hydrolytic activity and selectivity of the POMs depend on the structure of the protein substrate, as well as on the structure, polarity and charge of surfactants.…”

“…[9] Zr(IV)-substituted Keggin-type POM ((Et2NH2)10[Zr(α-PW11O39)2]; Zr-K 1:2) was used to promote the hydrolysis reaction since it has been previously shown to be active in the hydrolysis of several water-soluble proteins (Figure 1 (b)). [22][23][24] Results and Discussion…”