“Coiled coils for chloride indicators and other pursuits in protein modelling and design”

The design of proteins, i.e. finding an amino acid sequence that folds in a predetermined structure, has evolved from what was once considered an impossible task to a powerful tool for the realisation of protein architectures with novel structures and functions. Here I will present my recent work on the design of a protein domain which undergoes conformational transitions upon chloride binding. Starting from existing trimers of α helices (so called coiled coils) with Cl- binding sites at their interface, putative sequences of monomeric three α-helix bundles were found using established protein design tools (Rosetta). The folding landscape and the stability of the most promising sequences were analysed based on protein-structure prediction methods and extensive molecular dynamics simulations. The availability of such Cl--sensitive domains could be exploited for engineering novel fluorescence-based reporters of Cl- concentration inside cells and tissues, by genetically fusing the designed sequences to suitably re-arranged fluorescent protein genes (such as in the Cameleon and GCaMP families of Ca2+sensors).