DEVELOPMENT OF BIOSENSORS FROM NATURAL GREEN FLUORESCENT PROTEIN USING PROTEIN ENGINEERING APPROACH FOR HEAVY METAL DETECTION

We have applied protein engineering approach to develop biosensors for metal detection using Green Fluorescent Protein (GFP) expressed in natural jellyfish (Aequorea Victoria). Integrating with hard-soft acid base theory, we constructed a novel metal binding site in close proximity of the internal GFP chromophore by mutating amino acid residues involved in a hydrogen bonding network that facilitates the fluorescence intensity emission of the typical GFP-wild type. The first design, GFP-(Cys)₃, contains three mutated Cys generating a trigonal planar metal site within the GFP structure. The GFP-(Cys)₂, on the other hand, consists of two mutated Cys generating a linear metal site environment. It is hypothesized that when metal incorporates into the binding site it will perturb the fluorescence intensity of the designed biosensor. Fluorescence spectroscopy showed that both of the GFP-(Cys)₃ and GFP-(Cys)₂ are specific towards Hg(II) and Ag(I) as observed by a significant decrease in fluorescence intensity upon metal titration. The Ligand-to-Metal-Charge-Transfer (LMCT) in UV-VIS spectroscopy at ~236-239 nm (S ® Hg(II)) and ~238-240 nm (S ® Ag(I)) confirmed the presence of the metal-Cys interactions when the GFP-(Cys)₃ and GFP-(Cys)₂ binds with Hg(II) and Ag(I), respectively. None of the designs; however, does not interact with Zn(II), Ni(II), Cr(III), Cu(II), Pb(II), and Cd(II) as indicated by the unchange in fluorescence intensity and the absence of LMCT formation when the metal concentration increases. Due to a lack in a metal binding environment, GFP-wild type do not result in a decrease in fluorescence intensity and the LMCT formation in the presence of any metal. This information strongly proves that the GFP-(Cys)₃ and GFP-(Cys)₂ biosensors only bind to Hg(II) and Ag(I) because of the preference between the soft metal center and soft ligand characters. Circular Dichroism (CD) verified that all the GFPs were not denatured by Hg(II) and Ag(I) as the biosensors are able to maintain their secondary β-sheet structure through the metal titration. Thus, our work suggests that protein engineering is a powerful approach that we can use to design alternative biosensors for Hg(II) and Ag(I) investigation. It is hoped that the knowledge gained can be further developed to achieve the maximum benefits for metal detection in the fields of medicine, agriculture, and industry in the future.