Self-Assembled Protein–Surfactant Nanoaggregates for Tunable Peptide Bond Hydrolysis by Polyoxometalate Nanoclusters

Abstract

The development of robust artificial proteases is of crucial importance for the study of proteins since natural proteases only retain their proteolytic activity under specific conditions. The presence of surfactants, which aid in solubilizing proteins and in probing their structure, is particularly detrimental to natural proteases. Therefore, artificial proteases that can function in the presence of surfactants are needed. Here, we report the hydrolysis of horse heart myoglobin (Mb) in the presence of a Zr(IV)-substituted Keggin polyoxometalate cluster (Et2NH2)8[{α-PW11O39Zr-(μ-OH)(H2O)}2]·7H2O (Zr–K 2:2) as an artificial protease and different surfactants: sodium dodecyl sulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zw3-12), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The formation of nanoaggregates consisting of micellar structures containing the protein, the surfactant, and Zr–K 2:2 was detected by dynamic light scattering and conductivity measurements. Hydrolytic reactions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the hydrolytic efficiency was observed to increase in the presence of all three surfactants, but the fragmentation pattern was different depending on the nature of the surfactant used. Furthermore, a multitechnique approach combining cyclic voltammetry, 31P nuclear magnetic resonance, fluorescence, circular dichroism, and UV–vis spectroscopy was used to gain a better understanding of the protease activity of Zr–K 2:2 in the presence of surfactants. Based on this approach, a general model for the interactions typically observed in protein/surfactant/POM ternary nanoassemblies has been proposed. The hydrolytic efficiency of a POM nanocluster toward a protein in the presence of surfactants was found to depend on (i) the structure of the protein and accessibility of the cleavage sites, (ii) the structure of the surfactants, (iii) the ease of exchange between the POM and the surfactants on the surface of the protein, and (iv) the influence of surfactants on the speciation of the POM catalyst.

Exploring the Reactivity of Polyoxometalates toward Proteins: From Interactions to Mechanistic Insights

“…82,88 Moreover, it was shown that the hydrolytic activity of [Zr(P 2 W 17 O 61 ) 2 ] 16-(Zr-WD 1:2) toward the dipeptide glycyl-L-histidine was preserved in the presence of anionic, neutral, or zwitterionic surfactants as the surfactants did not affect the binding of the dipeptide to the M-POM. 88 Furthermore, in the presence of surfactants, M-POMs have also been reported to be hydrolytically active toward several globular and water-soluble proteins, 85,86,89,90 as well as toward unstructured and insoluble proteins. 87,91 The hydrolytic activity of M-POMs toward proteins in the presence of surfactants has been determined to depend on: (i) the protein structure and pI, (ii) the structure of the surfactant (charge, size and polarity), (iii) the ease of exchange between the surfactant and the M-POM on the protein surface, as well as (iv) the influence of the surfactant on the speciation of the M-POM (Figure 5a).…”

“…Unlike natural proteases, which lose their activity in the presence of surfactants, it has been shown that M-POMs are still hydrolytically active in surfactant solutions. , This is highly important since surfactants can be used to probe the structure of proteins . Additionally, surfactants are typically used to dissolve poorly soluble proteins, such as membrane proteins, that are often underrepresented in proteomics studies despite their important roles in many biological processes .…”

“…The hydrolytic activity of M-POMs toward proteins in the presence of surfactants has been determined to depend on: (i) the protein structure and pI, (ii) the structure of the surfactant (charge, size and polarity), (iii) the ease of exchange between the surfactant and the M-POM on the protein surface, as well as (iv) the influence of the surfactant on the speciation of the M-POM (Figure a) . The previously established ability of zwitterionic surfactants to interact with M-POMs can disrupt the equilibria between the dimer and monomeric forms of the M-POMs, which can negatively affect the hydrolytic activity (Figure a) . In addition, the hydrolytic reaction depends on the ability of the M-POM catalyst to exchange with the surfactant on the protein surface.…”

“…The importance of the ease of exchange between M-POM and surfactant on the protein surface is also reflected in the decrease in hydrolytic efficiency of M-POMs upon increasing the concentration of the surfactant. Moreover, this effect of the surfactant concentration was observed across widely different classes of proteins: HSA (a globular soluble protein), β-casein (an unstructured partially soluble protein) and zein (an insoluble protein that requires surfactants to be dissolved). ,, However, under the right conditions, the addition of surfactants can even be exploited to achieve significantly higher hydrolytic efficiencies or cleavage of additional sites due to partial unfolding of the protein structure, which can make cleavage sites more susceptible to undergo peptide bond hydrolysis catalyzed by the M-POM. ,, …”

“…These studies showed that cationic surfactants induced precipitation while neutral or anionic surfactants do not interact with M-POMs. , Interestingly, M-POMs were observed to interact with the positively charged ammonium groups of zwitterionic surfactants without inducing precipitation, resulting in M-POM incorporation into the micellar structures. , Moreover, it was shown that the hydrolytic activity of [Zr­(P 2 W 17 O 61 ) 2 ] 16‑ (Zr-WD 1:2) toward the dipeptide glycyl- l -histidine was preserved in the presence of anionic, neutral, or zwitterionic surfactants as the surfactants did not affect the binding of the dipeptide to the M-POM . Furthermore, in the presence of surfactants, M-POMs have also been reported to be hydrolytically active toward several globular and water-soluble proteins, ,,, as well as toward unstructured and insoluble proteins. , …”