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De novo Metallonucleases Based on Helix–Loop–Helix Motifs
Author(s) -
Rossi Paola,
Tecilla Paolo,
Baltzer Lars,
Scrimin Paolo
Publication year - 2004
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200400160
Subject(s) - chemistry , helix (gastropod) , circular dichroism , stereochemistry , crystallography , protonation , helix bundle , cooperativity , nucleophile , metal ions in aqueous solution , peptide , ligand (biochemistry) , metal , protein structure , catalysis , ion , biochemistry , organic chemistry , receptor , biology , ecology , snail
Three new 42‐mer peptides (PR I–III) designed to fold into a hairpin helix–loop–helix motif have been prepared. In the peptide sequence two (PR II–III) or four (PR I) copies of an unnatural amino acid bearing a triazacyclononane metal‐ion binding site (ATANP) have been inserted in appropriate positions to allow the ligand subunits to face each other either within the same helix or between the two helices of the hairpin motif. Circular dichroism (CD) studies in solution have shown that the apopeptides adopt a well‐defined helix–loop–helix tertiary structure that dimerizes in solution at concentrations above 200 μ M to form a four‐helix bundle. However, the helical content is strongly dependent on pH and metal‐ion binding. Both protonation of the amines of the triazacyclononane units present in the ATANP lateral arm and complexation with Zn II ions cause a significant decrease of the helical content of the sequences. The Zn II complexes of the three peptides catalyze the transesterification of the RNA model substrate 2‐hydroxypropyl‐ p ‐nitrophenyl phosphate (HPNP) with different efficiency. The best catalyst appears to be PR I–4 Zn II , that is, the peptide incorporating four ATANP units. Michaelis–Menten saturation kinetics allowed us to estimate that substrate fully bound to the catalyst reacts 380 times faster than in its absence. The kinetic evidence suggests cooperativity between (at least two) metal ions: one activating the nucleophilic species (directly or indirectly) and the other facilitating nucleophilic attack by coordination of the phosphate.