DESIGNING A PROTEIN-BASED BIOSENSOR FOR METAL DETECTION USING PROTEIN ENGINEERING APPROACH

We have integrated protein engineering strategy with bioinorganic chemistry principles to design biosensors for metal sequestration using a natural jellyfish Green Fluorescence Protein (GFP) scaffold. In this regard, we have altered amino acid residues located inside of the b-barrel that are involved in a hydrogen bonding network with the internal GFP chromophore with a purpose to generate a metal binding site within the GFP. The replacement of those native residues with cysteine (Cys) results in a thiolate binding pocket that is suitable for a soft metal binding.  We have engineered two biosensors. The first design, GFP-(Cys)₂, contains two positions of amino acids mutated to Cys. The second design, GFP-(Cys)₃, contains three mutations. From fluorescence spectroscopy studies, it was shown that both GFP-(Cys)₂ and GFP-(Cys)₃ are specific toward mercury [Hg(II)]  and silver [Ag(I)].  The intensity of the green fluorescence significantly decreases when the metal concentration increases. The GFP-wild type, on the other hand, does not bind to the metals as suggested by the unchange of fluorescence in the presence of metal. Thus, both of the deigns bind to Hg(II) and Ag(I) because of the preference between the soft metal and soft ligand characters. Circular dichroism spectroscopy confirms that the incorporation of the metal does not alter the secondary structure of the designed biosensor. This strongly indicates that our designed biosensors are capable of detecting such metals due to the change in their fluorescence capacity but simultaneously are able to remain their structural stability. Thus, our work has suggested that protein engineering is a powerful approach that we can employ to innovate alternative biosensors useful for Hg(II) and Ag(I) detection. These biosensors are protein-based and soluble in water, which can be further applied to use in vivo for future medical treatment and/or toxic metal determination for industrial and agricultural purposes.