Using coordination chemistry concepts for computational metalloprotein design

Protein design is being pursued for two important reasons: to test our knowledge about the protein structure-function relationship and to generate proteins with biotechnological applications ranging from industrial catalysis to biomedical engineering. The work presented here describes the computational metalloprotein design using concepts in coordination chemistry.;In one experiment, attempts were made to design a rhodium-binding site into the cavity of intestinal fatty acid binding protein (IFABP). The geometric definition for the binding site was derived from the hydrogenase metal complex which resembles the Cys2Met2 coordination sphere. These protein designs were constructed using recombinant DNA technology. The metal binding was studied using UV-Vis spectroscopy taking advantage of the metal-thiolate charge transfer bands. The designed proteins did not bind to the metal ions such as Rh3+, Cu2+, and Ni2+ ions because of their small size. However a stoichiometric binding was observed with larger metal ions such as Pd2+ and Pb2+ ions. These studies suggested the rigidity of the beta-sheet secondary structure motif of IFABP.;In another experiment, a phosphate binding protein was converted into a lead binding protein (termed PbBP) using the coordination chemistry of Pb 2+ ions. This PbBP is then developed as Pb2+ ion biosensor. The reagentless biosensor developed here was based on the previously developed strategy for maltose sensing using maltose binding protein (MBP), redox active ruthenium complex, and semiconducting nanoparticles. This extension of maltose sensing to Pb2+ ion sensing demonstrated that the structurally similar proteins can be swapped with MBP to generate biosensors for other analytes. The Pb2+ ion biosensor developed here was highly selective, sensitive (500 pM lower limit of detection), and reproducible even in the presence of 10 mM Ca2+ ion (major competitor for Pb 2+ ions in blood). It was shown to respond with red-emitting and -absorbing InGaP ZnS nanoparticles allowing their application for detection in red blood cells. The biosensor was then shown to respond to Pb2+ ions in 1% red blood cells.