Protein prosthesis: β-peptides as reverse-turn surrogates

Abstract

The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic β-dipeptide: β²-homoalanine-β³-homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.

Figure 1

Ribbon diagram of wild-type RNase A (PDB entry 7rsa6) and structures of reverse turns relevant to this work. The number of atoms in the hydrogen-bonded rings of each artificial turn is indicated above the hydrogen bond. The Asn113–Pro114 peptide bond of wild-type RNase A is in the cis conformation.

Figure 2

Thermally induced transition of β²β³hAla RNase A (open symbols) and the wild-type enzyme (closed symbols). Unfolding was followed by CD spectroscopy at 278 nm as described in the Materials and Methods section.

Figure 3

Gdn–HCl-induced transition of β²β³hAla RNase A (open symbols) and the wild-type enzyme (closed symbols). Unfolding was followed by CD spectroscopy at 278 nm as described in the Materials and Methods section.

Figure 4

Arrhenius plot of the unfolding rate constants of β²β³hAla RNase A (open symbols) and the wild-type enzyme (closed symbols). Values of k_U were determined by limited proteolysis with thermolysin as described in the Materials and Methods section.