Redesign of SpyTag peptide toward artificial stereoselective metalloenzymes: copper binding and catalysis

Metalloproteins promote some of the most complex biomolecular processes in nature. Here we the redesign of the Spy protein toward an artificial copper protein. The Spy construct 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 1a). 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 synthesis also introducing non-natural amino acids, thus expanding the space of redesign of the Spy protein toward applications in industry, biotechnology and nanotechnology. Figure 1: a) Representation of SpyTag (red) and SpyCatcher (blue) along with the SpyTag sequences studied. The (His)2 site is represented in green; b) Model of the Cu(II)/(His)2 site. SypTag peptides bearing a bis-histidine site were prepared. These peptides are known 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. Spectrophotometric experiments to study the oxidation of 4-methylcatechol and L- /D-DOPA were carried out to assess the possible presence of stereoselectivity among the latter two substrates (Figure 1b).