Uncontrolled hydrolysis is the ultimate enemy when trying to work around the unwanted aqueous chemistry of abundant metals like titanium and vanadium. These metals, often used in industrial settings, are not seen much in biological environments because of their propensity to undergo this uncontrolled hydrolysis. However, nature has found ways around unwanted hydrolysis in many metals, including stabilizing iron. Using this as a model, a metalloprotein system has been developed that stabilized Ti and V in an aqueous setting, thus functionalizing them to cleave DNA. The Due Ferri single chain (DFsc) protein system has two iron binding sites and was modeled from natural proteins. The main focus of this project is biophysically characterizing the metal binding of the DFsc proteins with tyrosine added to the active site (DY, DFY, G4Y) to better understand the structure function relationships in these enzymes. Furthermore, we are curious if the substitution of tyrosine improves metal stabilization and the binding interactions of the DF variants compared to proteins without tyrosine. We focused on titanium, vanadium, and zinc binding to the proteins, and were able to monitor the secondary structure of the protein associated with metal binding by using circular dichroism spectroscopy. Direct measurement of metal binding using inductively coupled plasma – optical emission spectroscopy will be used to confirm binding stoichiometry. Initial results indicate that the tyrosine addition may be an important factor in stabilizing metal-protein interactions in the DFsc system.

Biophysical characterization of metal binding to <i>de novo</i> due ferri proteins

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Biophysical characterization of metal binding to de novo due ferri proteins