| Here I describe the application of rational protein engineering of bacterial periplasmic binding proteins to the design of thermostable biosensors. Periplasmic glucose- and ribose-binding proteins from thermophilic organisms have been identified, cloned and characterized. The structure of periplasmic glucose-binding protein from Thermotoga maritima (tmGBP) has been solved to 1.7 A resolution by X-ray crystallography. A series of robust glucose biosensors have been constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions responsive to ligand-induced conformational changes. Among tmGBP-based glucose biosensors, the Y13C•Cy5 conjugate signals strongly and responds to glucose concentrations appropriate for in vivo monitoring of blood glucose levels (1-30 mM). The Y13C•Cy5 conjugate has been immobilized onto microtiter plates in both semi-specific and orientation-specific manners to give reversible responses to glucose. The immobilized protein also retains its response after long term storage at room temperature.;In addition, E. coli ribose-binding protein (ecRBP) has been stabilized by rational protein engineering to enhance its suitability as a scaffold protein for use in computational design. Several approaches have been exploited to improve the thermostability of ecRBP, including the introduction of mutations to decrease the entropy of the unfolded form, the replacement of un-favored polar amino-acids in the protein core with non-polar residues, the engineering of disulfide bonds, and the incorporation of features from thermophilic RBPs. The stabilizing mutations achieved from these approaches were evaluated individually and then combined in a stepwise manner, resulting in a variant with a melting temperature 17.5°C higher than ecRBP, which can also serve as a stable scaffold protein for biosensor design. |
