REDESIGN OF SPY SYSTEM INTO ARTIFICIAL METALLOENZYMES: COPPER BINDING AND CATALYSIS

Metalloproteins promote some of the most complex biomolecular processes in nature. The interest in the development of new metal proteins and artificial enzymes provided the basis for the development of the Protein Design. The project presented here relates with the redesign of the Spy protein. This 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 (Figure 1). On one hand the peptidic part 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 also introducing non-natural amino acids, thus expanding the space of redesign of the Spy protein toward applications in industry, biotechnology and nanotechnology. We studied SypTag peptides bearing bis-histidine sites, 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 catechols (4-methylcatechol and DOPA) in the presence of the Cu(II)/peptide adducts was performed. The design of a second generation of peptides bearing a His-His site will be presented and discussed.