Sequence-Selective Protection of Peptides from Proteolysis

Abstract

Proteolysis of proteins and peptides is involved in the infection of cells by enveloped viruses and also in the invasion and spread of cancer cells. Shutting down broad-specificity proteases, however, is problematic because normal functions by these proteases will be affected. Herein, nanoparticle receptors were prepared from molecular imprinting for complex biological peptides. Their strong and selective binding enabled them to protect their targeted sequences from proteolysis in aqueous solution at stoichiometric amounts. Generality of the method was demonstrated by the protection of hydrophobic and hydrophilic peptides from different proteases, selective protection of a segment of a long peptide, and selective protection of a targeted peptide in a mixture. Most interestingly, two receptors targeting different parts of a long peptide could work in cooperation to protect the overall sequence, highlighting the versatility of the method.

Keywords: inhibition; molecular imprinting; peptide; protease; protection.

Scheme 1

Preparation of peptide‐binding MINP from molecular imprinting of a cross‐linked micelle.

Figure 1

a,b) Proteolysis of A‐III in 10 mm phosphate buffer (pH 7.4) under different conditions by a) trypsin and b) chymotrypsin. c,d) Correlation between the binding free energies of MINPs for A‐III and the protection factors of the MINPs in proteolysis by c) trypsin and d) chymotrypsin. Blue data point was for the full sequence, green points were for the N‐terminal sequences, and red for the C‐terminal sequences. e,f) Nonlinear least squares curve fitting of the conversion of A‐III by e) trypsin and f) chymotrypsin digestion in 10 mm phosphate buffer (pH 7.4).

Figure 2

Product distribution curves in the trypsin digestion of Aβ_1–28 in a) buffer and in the presence of 1 equiv b) NINP, c) MINP(β_1–14), and d) MINP(β_15–28).

Figure 3

HPLC chromatograms of trypsin digestion of a 2:1 mixture of Angiotensin III and Aβ_1–28 by a) trypsin without any protection, and in the presence of b) MINP(A), c) MINP(A) & MINP(β_15–28), and d) MINP(β_1–14) & MINP(β_15–28). Reaction time was 4 h except in (c) which required 12 h for the selective hydrolysis of Aβ_1–28.