| This work describes the application of engineering protein-ligand interactions to the design of biosensors and multisensors. Structure-based computational design was used to engineer a zinc binding site in the enzyme ATPase. As a result, zinc acts as an allosteric regulator of the enzymatic activity. Computational design was further applied to the redesign of the binding specificity of glucose- and ribose binding proteins to bind pinacolymethylphosphonic acid (PMPA), a degradation product of the nerve agent soman. The computationally redesigned binding proteins were labeled with a thiolreactive fluorophore at a unique cysteine position and as a result, a change in fluorescence is exhibited by the protein-fluorophore conjugate in response to ligand binding. The results demonstrate that the engineered proteins act as reagentless fluorescent biosensors for PMPA and exhibit a range of affinities between 0.045 and 10 muM. Protein engineering techniques were used to extent the ability of a single biosensor element to distinguish between several similar target ligands by incorporating many sensor elements in a multisensor system. The protein PhnD, a periplasmic binding protein that binds many phosphonates, was characterized, and variants were constructed by introducing point mutations in its binding pocket. The PhnD variants exhibit differential binding affinities to several similar molecules and were used as sensor elements in a fluorescent multisensor system. The multisensor can be used to determine the concentrations of many analytes in a solution and can detect the presence of an interferent for which it has not been characterized by taking advantage of the non-linear nature of the fluorescent response to ligand binding. |
