Humboldt-Universität zu Berlin - Experimentelle Biophysik

Nicolas Liem

In Jan 2020, I began my research on the investigation of rhodopsin phosphodiesterases. These membrane-bound enzymes carry a retinal cofactor which is essential for photoswitching of the enzyme. The enzyme breaks down cyclic GMP depending on the illumination with blue light [1]. This has significant medical relevance because various physiological functions are under the control of cGMP. Optogenetic PDEs can be a novel methodical tool to dissect cyclic nucleotide signaling, assisting the search for new therapeutic targets by probing cyclic nucleotide signaling at unprecedented spatiotemporal resolution.

To characterize and engineer these enzyme rhodopsins, I am expressing this protein in mammalian cells (such as HEK293T, ND7/23), and in the yeast P.pastoris. Various affinity-tag systems are then used to purify the protein and to characterize it spectroscopically. To test the enzymatic function and its light-dependency, I am focusing on HPLC methods. Eventually, these approaches have been fruitful and allowed functional measurements in electrophysiology, where bidirectional optogenetic control over CNG channels was achieved with a rhodopsin cyclase / rhodopsin phosphodiesterase combination [2]. To address this direction of spectral multiplexing, I am relying on protein engineering as well as setup modifications using new photonic devices. Further activities include teaching undergraduate practical courses in optics and improving their respective setups.

[1] Brunet, T., et al. (2019): "Light-regulated collective contractility in a multicellular choanoflagellate", Science 366(6463):326-334