(BBSRC DTP) Understanding protein dynamics in phytochromes: redesigning important photoreceptors with non-native amino acids

The recent advances in using non-natural amino acids to expand upon Nature’s genetic code have highlighted the potential to revolutionise our understanding of structure-function relationships in proteins. In particular, this state-of-the art approach provides new and highly attractive routes for understanding the role of conformational changes in proteins, which would otherwise prove impossible by using standard protein engineering methods. It is also now well known that proteins are inherently dynamic molecules, exhibiting motions on timescales ranging from bond stretches (fs) through to slow domain motions and global structural changes (ms), all of which are essential for their function. One such family that use this whole range of protein dynamics for their activity are the phytochromes, which are photoreceptors that regulate a range of responses in plants and cyanobacteria. They have huge potential in synthetic biology programmes and offer new and exciting opportunities in the field of optogenetics. However, to rationally engineer these proteins as light-responsive ‘Parts’ for use in synthetic biology applications we need to develop a detailed understanding of the structural transitions that underpin light-induced signalling.

Phytochromes are bilin-containing photoreceptor proteins that photoconvert between a red light-absorbing (Pr) state to a far-red light-absorbing (Pfr) state. Photoconversion between the Pr and Pfr states involves photoisomerisation of the bilin cofactor, which is coupled to large-scale conformational changes in the protein. However, the lack of a full-length crystal structure has limited our understanding of how light-driven chemical changes drive the protein structural changes that are essential for phytochrome function. The student will overcome this by using non-natural amino acids to produce modified phytochrome proteins that contain genetically encoded fluorescence probes and nitroxide spin labels at defined positions throughout the protein. This will allow a series of inter-label distances to be measured, which will facilitate the determination of any conformational changes during the photocycle of the phytochrome. This is a highly interdisciplinary project at the cutting edge of protein engineering research and will provide the student with expertise in new molecular biology methods, protein expression / purification, biochemical assays, laser spectroscopy and EPR techniques.

https://sites.google.com/site/scruttonlab/

Further Information
For more information and informal inquiries please contact Dr Anthony Green: [Email Address Removed]