Engineering of bacterial periplasmic binding proteins to change selectivity and increase signal-to-noise ratio

Understanding the distribution of complex organic molecules associated with living things is essential to answering the questions expressed in the Astrobiology Roadmap that are “How does life begin and evolve?” and “Does life exist elsewhere in the universe?” The objectives of this proposal are to change the selectivity of periplasmic binding proteins (PBPs) and to increase signal-to-noise (S/N) ratio in order to develop a new technology for measuring the abundance of an array of organic molecules, with stereochemical specificity, in complex mixtures, without chemical modification. In our previous work, we have developed various Fluorescence Resonance Energy Transfer (FRET) based protein biosensors from thermophilic bacteria which are very specific for their respective L-isomers (Paavola et al, 2008) with S/N ratios between 5 and 10. However, higher S/N ratios (>20) are desirable to reduce the cost and complexity of the required optical hardware. In order to achieve higher S/N ratios, this proposal will employ Luminescence Resonance Energy Transfer (a variation of FRET) technique and to emulate the behavior of the PBP's chemotactic system. To change the selectivity, the latest computational protein design and genetic engineering techniques will be employed to produce variants of PBPs that will specifically bind to the molecule's D-isomer.