Recombinant Protein Based Ca+2Ion Sensor Designing; an In-Vitro Test of Folding Coupled to Binding Hypothesis

Recombinant protein-based sensor designing is a burgeoning topic of interest due to its potential in-vivo applications. Proteins are particularly suitable for sensor designing as they have high cell viability, negligible cytotoxicity, interaction with the substrate with a large three-dimensional surface and binding with high affinity. But protein-based sensors are scarcely available as engineering protein as a functional sensor is very challenging. I will describe the designing of a recombinant protein-based Ca+2 ion sensor using “folding coupled to binding” strategy. The Ca+2 ion binding domain of a natural protein is used as a template and site-selective mutations are utilized to destabilize the protein structure and couple its folding-unfolding kinetics with the ligand association equilibrium. In the coupled equilibrium, the apo (Ca+2 free) form of the protein folds only in presence of the target analyte and the structural changes in the protein is exploited as a transducer for the biosensor. We have utilized both FRET and PET-based techniques to estimate the ligand-induced changes in protein structure. To probe less than 2 nm distance change between folded and unfolded states of the protein, photo-induced electron transfer (PET) is exploited as an alternative to FRET. An organic fluorophore is covalently attached to one site of the protein and at another site, a selected amino acid is mutated to tryptophan. In folded state (Ca+2 bound) the intramolecular electron transfer from the tryptophan to the fluorophore resulted in a low fluorescence, which increased over 4 times in the unfolded state (Ca+2 free) of the protein, resulting in a large change in the fluorescence signal. The successful in-vitro detection of Ca+2 ions confirms the eligibility of the “folding coupled to binding” strategy as the next generation sensor designing methodology.