Bottom-Up Design of Synthetic Photoactive Metalloproteins

This dissertation describes the design of synthetic photoactive metalloproteins and their metal binding properties. Four peptides are prepared to study the interplay between peptide backbone and metal ion clusters. The four peptides synthesized are: K IEALEGK CEACEGK IEALEGK-GGY mini-C9C12-GGY Q IAALEQK IAALEQK CAACEQK IAALEQK GGY AQC16C19-GGY Q IAALEQK IAAVEQK CAACEQK IAALEQK GGY AQVC16C19-GGY Q IAAVEQK CAACEQK IAALEQK GGY miniAQVC16C19-GGY Starting from a previously designed peptide C16C19-GGY, a smaller peptide, mini- C9C12-GGY was designed. Mini-C9C12-GGY has the same peptide sequence with C16C19- GGY except that it has less amino acids than C16C19-GGY at N-terminus. It was shown that mini-C9C12-GGY also can bind to Cu(I) to form a tetrameric metalloprotein Cu(I)/mini-C9C12-GGY which displays an intense room-temperature luminescence around 600 nm. It was also shown that the Cu(I) coordination in Cu(I)/mini-C9C12-GGY might change at high loading of the Cu(I). The emission of the Cu(I)/mini-C9C12-GGY follows biexpotnetial decay and can be quenched by a series of [Ru(NH3)5X] (X= chloro, amine, lutidine, pyridine, nicotinamide, and 3,5-dimethyl pyridine dicarboxylate). The quenching mechanism is assigned to a photoinduced electron-transfer event by transient spectroscopy. The bimolecular electron-transfer of Cu(I)/mini-C9C12-GGY occurs in the diffusion limit region. Such results support our hypothesis that the hydrophobic condition provided by the coiled coil structure in Cu(I)/C16C19-GGY results in the prohibition of close approach between the donor and acceptor. The effect of the stability of the apopeptide folding on the oligomerization states of the metalloprotein was demonstrated by a series of peptides derived from a dimeric peptide motif: Q(IAALEQK)nGGY (n\u3e3). The peptides are AQC16C19-GGY, AQVC16C19GGY, and miniAQVC9C12-GGY. AQC16C19-GGY and AQVC16C19GGY exists as coiled coil at their apo-states. MiniAQVC9C12-GGY exists as a random coil due to the short peptide sequence compared to AQC16C19-GGY and AQVC16C19GGY. Although the peptides have different free energies of folding at their apo-states, all the resulting metalloproteins after the addition of Cu(I) exist as trimers as determined by ultracentrifugation analysis and/or high performance size exclusion chromatography. These results indicate that metal ions are still the dominating factor in determining the oligomerization structures of metalloproteins in the researched system