Engineering Protein Scaffolds for Ultrafast, Broad Range, Single-Molecule Analog pH Biosensors

The research described in this thesis aims at exploiting recent important theoretical and experimental developments in protein folding towards developing advanced biosensors with improved/novel properties. Transforming this basic protein folding knowledge into engineering strategies for designing novel biosensors is an enormous challenge. Here we demonstrate the very first steps in that direction.Biosensors based on proteins that naturally toggle between two states (unfolded/folded) upon specific binding to a target molecule have been successfully used for real-time sensing. These devices behave as conformational switch sensors. Principles on how to engineer this type of conformational transducer onto any protein of interest have also been laid out. Our work takes this state of the art forward to design high-performance conformational rheostat sensors. The rationale is to develop sensors with expanded dynamic range and faster response time by using as conformational transducer the coupling of binding to the analyte and the gradual folding process of fast folding protein modules, and fluorescence as optical readout. As proof of concept, we investigate the pH sensing capabilities of engineered proteins based on two scaffolds: i) an anti-parallel coiled-coil, and ii) a tandem array of the small downhill folding domain BBL. Our results reveal that such pH sensors exhibit a linear response over at least 4 orders of magnitude in proton concentration. We also demonstrate that a pH sensor based on a conformational rheostat transducer can produce analog pH readouts at the single-molecule level together with ultrafast response (< 1ms). These results lay the ground for the development of fluorescence biosensors for the analog monitoring of pH in real time and with nanoscale spatial resolution. Finally, we introduce a platform for the plug and play implementation of fully genetically encoded fluorescence conformational biosensors