Highly Selective and Tunable Protein Hydrolysis by a Polyoxometalate Complex in Surfactant Solutions: A Step toward the Development of Artificial Metalloproteases for Membrane Proteins

Abstract

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.

Figure 1

(a) Equilibria between the 1:2, 1:1, and 2:2 species of the Zr(IV)-substituted Wells–Dawson POM. WO₆ octahedrons are represented in blue, PO₄ tetrahedrons in red, and Zr(IV) in green. (b) Chemical structure of the zwitterionic surfactant, CHAPS.

Figure 2

Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels of HSA hydrolysis by Zr1-WD2 POM in phosphate buffer (10 mM, pH 7.4) at 60 °C in the absence and presence of surfactants: Influence of increasing % w/v CHAPS after 2 days of incubation. From left to right: protein ladder; 0.0, 0.2, 0.5% CHAPS; protein ladder; 1.0, 2.0% CHAPS.

Figure 3

3D structure and surface charge distribution. Negatively charged surface areas are shown in red, and positively charged surface areas are shown in blue. Asp residues in Asp–X bonds that are hydrolyzed are shown in yellow. The image was created using PyMol molecular visualization system software.

Figure 4

CD spectra of HSA (5 μM) in phosphate buffer (10 mM, pH 7.4, 10% D₂O) in the absence and presence of increasing % CHAPS.

Figure 5

Emission fluorescence spectra of HSA in the presence of different concentrations of Zr1-WD2 ([HSA] = 10^–5 M, [CHAPS] = 0.5 wt %, pH = 7.4). From top to bottom, the concentration of Zr1-WD2 was increased stepwise from 0 to 10^–5 M with increments of 10^–6 M.