Reagentless Fluorescence Sensors Produced by Protein Engineering

Abstract

This chapter describes reagentless fluorescence sensors produced by protein engineering. The measurement of ions such as H+ and Ca2+ with fluorescent indicators is well established and they have been used both with optical fiber sensors, and with fluorescence microscopy for intracellular measurements. An alternative strategy that is to modify known binding proteins with a specifically located fluorescent reporter group, whose properties change between the ligand-free and ligand-bound forms has been adopted. The key to this approach is the site-specific placement of a fluorescent reporter group, and hence conventional chemical labelling is totally inadequate in this context. The protein chosen to test this approach is the maltose binding protein. Mutants of this protein with a cysteine residue in the region where the structural change occurs were prepared, and labelling was done of the mutants with polarity sensitive fluorescent probes such as nitrobenzoxadiazole or dansyl derivatives and used the resulting molecules to measure maltose concentrations in the micromolar region.