Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

54-POS Board 54 Developing Genetically Encoded Actuators with an Engineered Photocleavable Protein, PhoCle Wei Zhang , Robert E. Campbell. University of Alberta, Edmonton, Canada. Here, we report a photocleavable protein (PhoCle) engineered in Campbell Lab from green-to- red photoconvertible fluorescent protein, mMaple. PhoCle is a circularly permutated variant of mMaple. Like all known green-to-red photoconvertible fluorescent proteins, PhoCle can be converted by violet light (~400 nm). This photoconversion involves a covalent bond break of peptide backbone at the chromophore. Upon photoconversion, PhoCle spontaneously dissociates into two fragments. Improved variants of PhoCle are engineered by using directed evolution. Genetically encoded actuators are being developed with PhoCle. One strategy is to combine PhoCle with the well-established approach of conditional inactivation of protein by genetic fusion to a steroid receptor (SR). Briefly, the protein-of-interest is fused to SR via a PhoCle linker such that it is in an inactive (caged) state until it is cleaved away from SR by illumination. Using this approach we have demonstrated photo-uncaging of Cre recombinase and the Gal4- VP16 transcription factor. Another strategy is activating protein by PhoCle-mediated release of a tethered inhibitory domain. PhoCle-caged protease has be developed by using this approach. Overall, PhoCle shows its potential to be a powerful and versatile new technology to develop genetically encoded actuators.

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