Platform 1 will combine genome mining and protein engineering with automated plasma
membrane expression screening and automated photocurrent measurements to develop
optogenetic actuators for the proposed optogenetic therapies. The substantial dataset
collected under standardized conditions will furthermore expedite opsin fitness
landscape mapping and serve as a foundation for data-driven protein engineering
approaches, which will complement structure-guided approaches, thereby accelerating the
advancement of the next generation of optogenetic actuators.
Figure: Opsin Engineering Platform. In close collaboration with
all EKFZ Platforms and Teams, the opsin engineering platform (Platform 1) will
engineer improved ChRs and opto-GPCRs to combine maximal efficacy with minimal risk
of adverse effects. We will generate opsins with a red-shifted action spectrum,
plasma membrane localization, hypoimmunogenicity and suitable kinetics. (A-B)
Platform 1 will employ genome mining and protein engineering to develop next
generation optogenetic actuators for future optogenetic therapies. Shown are
ClustalW alignments and a phylogenetic tree used to identify new natural ChRs
(A) and the high resolution structure of a ChR (ChR2 structure, PDB ID: 6EID)
used to identify amino acid likely to improve the properties. We will employ 3D
models (AlphaFold, DeepMind) and cryo-EM analysis (Titan Krios G4, cryo-EM platform
of the University of Göttingen) to gather structural information in cases where high
resolution structures are not yet available (B). (C-D) The application
of automated procedures enables large scale screenings of libraries comprising the
found natural opsins and proposed opsin mutants. The established pipeline consists
of the spinning-disk-microscopy based CellVoyager CV8000 (Yokogawa), which is used
for automated opsin plasma membrane expression screenings (C), and the
Opto-SyncroPatch 384 (Nanion) for automated photocurrent measurements (D), to
analyze ChR properties. (E) Exemplary ChReef photocurrent, measured on the
Opto-SyncroPatch 384 at −60 mV. The substantial dataset collected under standardized
conditions will furthermore expedite opsin fitness landscape mapping and serve as a
foundation for data-driven protein engineering approaches which will complement
structure-guided approaches, thereby accelerating the advancement of the next
generation of optogenetic actuators.
