Humboldt-Universität zu Berlin - Experimentelle Biophysik

Nicolas Liem

With a fellowship from the BIG-NSE/Einstein center for catalysis, I started my PhD at Hegemann lab. 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.

I characterized and engineered these enzyme rhodopsins, applying and optimizing a range of wetlab techniques and spectroscopy methods:

• Cloning of point mutants and bicistronic constructs
• Expression of the full-length protein in HEK293T cells, ND7/23 cells, and in the yeast P.pastoris.
• Large-scale expression in P.pastoris, solubilization, and affinity-tag purification
• Protein science and protein quality control - SDS/PAGE, immunoblotting, size-exclusion chromatography, UV/vis spectroscopy (stationary/time-resolved), fluorescence spectroscopy. 
• Development of functional assays in a 96-well plate format, optimization of sensitivity and robustness in ELISA and HPLC-based detection
• Development, transfer, and validation of reverse-phase HPLC methods for the ratiometric and absolute quantification of purine and pyrimidine nucleotides, caretaking of the HPLC device.
• Translation to a functional electrophysiology experiment, achieving bidirectional optogenetic control over CNG channels [2]. Optimization of spectral multiplexing by protein engineering and setup adaptation using new photonic devices.

Furthermore, I am teaching undergraduate practical courses in optics. Here, I am improving the experimental kits for robust, hands-on results, I revise scripts for readability, and proofread student's protocols.

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