The objective of this research is to create a protein-based molecular sensor within the cell by inserting the "software"-- a synthetic gene, which encodes for the sensor -- into the "hardware" of the cell. Upon turning the gene on, the cell will produce functional sensor molecules, which will report upon an intracellular parameter of interest. The sensor consists of two, integrated components: an environmentally-responsive, elastin-like polypeptide (ELP), which can be designed via its amino acid sequence to be acutely sensitive to intracellular parameters, such as temperature, pH or ionic strength. Upon a change in the intracellular parameter, the ELP contracts and its end-to-end distance decreases by ~50%. The second component is a reporter, which converts this nanometer scale contraction of the ELP into a readout signal: a change in fluorescence. This is achieved by fluorescence resonance energy transfer (FRET) between -blue fluorescent protein (BFP)- the donor fluorophore, and - green fluorescent protein (GFP) - the acceptor fluorophore, each of which are fused at the gene level to opposite ends of the ELP. The central hypothesis of this proposal is that collapse of the ELP, in response to the altered concentration of an intracellular parameter of interest, will alter the distance between the donor (BFP) and acceptor (GFP) fluorophore, leading to enhanced FRET, and will thereby provide a detectable fluorescence signal. Two different physiological sensors will be fabricated and tested: a pH sensor, which incorporates ionizable residues in the ELP sequence, and a phosphorylation sensor, which contains a peptide substrate within the ELP. Altered pH or kinase concentration will isothermally induce the inverse transition, leading to fluorescence readout due to altered FRET. These prototype sensors will find application in fundamental cell and tumor biology studies, as well as in biotechnology applications, such as real-time, intracellular monitoring of industrial bioprocesses.
