Metal ions play a diverse and important role in some of the most chemically challenging reactions in biology. However, due to the many devastating diseases caused by metal ion imbalances, biological systems maintain strict control over metal ion concentration. The current methods for measuring metal ion concentrations in vivo involve fluorescent dyes from which it is difficult to obtain accurate measurements. For this reasons, protein-based metal ion biosensors would be beneficial. Large amounts of structural data on metalloproteins are now available die to advances in protein structure determination. A great deal about protein metal sites, such as metal coordination preferences and metal site geometry, can be obtained from these metalloprotein structures (http://metallo.Scripps.edu). As a result, knowledge of protein metal ion binding has greatly improved. If we can understand protein metal sites, we should be able to create them by design, testing our knowledge by experimentation. We have taken a recursive approach to rationally design a novel protein metal site within Green Fluorescent Protein (GFP). Our goal is to take advantage of the natural fluorescent properties of GFP in the creation of a metallobiosensor by linking metal ion binding to a spectroscopic change. Crystal structures of our family of GFP mutants show that zinc binds to the designed ligands and the site is preorganized for zinc binding. We now propose to alter the metal site to control metal ion specificity and affinity and to optimize spectroscopic changes induces by metal ion binding to the GFP designed site. The resulting GFP metal binding mutants would represent the next step in the design of proteins-based metal sensor that has the potential to be further optimized for in vivo metal ion detection, and they would have the potential to be great use both for sciences and for medicine.
