SpyTag peptides: focus on copper binding and stereoselective oxidase activity

Metalloproteins promote some of the most complex biomolecular processes in nature. We present here results related to the redesign of the Spy protein into an artificial metalloenzyme. The Spy protein consists of a peptide (SpyTag) that spontaneously binds to a protein (SpyCatcher) through an isopeptide bond, to give rise to a recombined Spy protein (figura1a). The reason of the choice of this construct for devising a new artificial metalloenzyme is two-fold. On one hand the peptide component of the protein may allow a straightforward introduction of metal binding sites on the final protein without redesigning the entire protein construct. On the other hand, peptides can be prepared by solid-phase synthesis also introducing non-natural amino acids, thus expanding the space of redesign of the Spy protein toward applications in industry, biotechnology and nanotechnology. We introduced on SypTag peptides a bis-histidine site, capable to bind Cu(II) and to promote reactions of oxidation of catechols. The affinity constants of the SpyTag peptides for Cu(I) and Cu(II) were determined by spectrophotometric and spectrofluorimetric titrations. Also, an evaluation of the kinetics in the oxidation of catechol, like 4-methylcatechol (figura 1b), in the presence of the Cu(II)/peptide adducts was performed. After that, DOPA in its two enantiomers was used to evaluate possible stereoselectivity in substrate oxidation.