Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: active sequence optimization.

Abstract In previous studies we showed that Ni(II) ions can hydrolytically cleave a peptide bond preceding Ser/Thr in peptides of a general sequence R N –(Ser/Thr)–Xaa–His–Zaa–R C , where R N and R C are any peptide sequences. A peptide library screening, assisted by accurate measurements of reaction kinetics for selected peptides, demonstrated the preference for bulky and aromatic residues at variable positions Xaa and Zaa [A. Krezel, E. Kopera, A.M. Protas, A. Wyslouch-Cieszynska, J. Poznanski, W. Bal, J. Am. Chem. Soc., 132 (2010) 3355–3366]. In this work we used a similar strategy to find out whether the next residue downstream to Zaa may influence the reaction rate. Using an Ac–Gly–Ala–Ser–Arg–His–Zaa–Baa–Arg–Leu–NH 2 library, with Zaa and Baa positions containing all common amino acids except of Cys, we found a very strong preference for aromatic residues in both variable positions. This finding significantly limits the range of useful Xaa, Zaa and Baa substitutions, thus facilitating the search for optimal sequences for protein engineering applications [E. Kopera, A. Belczyk-Ciesielska, W. Bal, PLoS One 7 (2012) e36350].