title: Design neuer rotlichtaktivierter Enzyme zur Kontrolle von zyklischen Mononukleotiden
Creators: name: Stabel, Robert
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: Optogenetics
subject: Photoreceptors
subject: Protein engineering
subject: 500

description: Sensory photoreceptor proteins mediate diverse light responses across organisms from all domains of life. Like other signal receptors, they feature modular architecture and comprise photosensor and effectors. The sensor module incorporates a chromophore to absorb light of distinct wavelength; conformational signals are then propagated via a linker to the effector to trigger a specific biological response. The modular composition enables the engineering of novel photoreceptors via rewiring of the sensor and effector modules. A particular challenge lies in the coupling of the modules such that productive signal propagation is maintained. Photoreceptors underpin the field of optogenetics where they enable the minimally invasive control of diverse processes in cells and organisms with high spatiotemporal resolution and exquisite molecular specificity. This thesis covers the engineering of synthetic photoreceptors for optogenetic deployment. To systematically probe the linker between sensor and effector modules, the PATCHY method is applied to two derivatives of the histidine kinase YF1. In the original lightrepressed YF1, linker lengths of 7n (n = 1, 2, 3, ...) and 7×n+1 are associated with lightrepressed and light-activated responses, respectively. For the two YF1 variants, lightregulated activity is also concentrated in discrete length registers, albeit with inverted signal response and to lesser extent. Linker length evidently governs output activity of the receptor and hence provides an efficient means for its rational modification. The main chapters of the thesis focus on the engineering of novel photoreceptors for the optogenetic control of cyclic mononucleotide metabolism. In the cell, phosphodiesterases (PDE) and nucleotidyl cyclases (ACs or GCs) control the level of cyclic mononucleotides which serve as second messengers in many important responses in eukaryotes and prokaryotes alike. This thesis expands the range of optogenetic tools by novel red/far-red- (R/FR) light-regulated cyclases and phosphodiesterases, thus allowing to study cellular metabolism with enhanced control. To this end, the light-inert sensor module of a cyclase is exchanged for different R/FR-switchable bacteriophytochrome (BphP) sensor modules to create novel photoactivated adenylyl cyclases (PACs). A fluorescence-based reporter assay enables the characterization and optimization of the PACs. The variant DdPAC, based on the Deinococcus desertii BphP, shows a 9.8 fold activity increase under red light and complete shutting off via far-red light. Likewise, the light-activated PDE LAPD is improved in terms of hydrolysis activity, regulatory efficiency and basal activity. To this end, the BphP and the PDE modules of LAPD are substituted for homologues photosensor and effectors. The variant Dr-BtPDE2A, based on the Deinococcus radiodurans BphP and the Bos taurus PDE2A, exhibits the best performance with an 8-fold increased activity upon red light and an improved off-switching upon far red-light illumination. For use in optogenetics, LAPD variants can be used alone or in tandem with PACs to manipulate cyclic mononucleotides levels and downstream processes in living cells like the gating of certain ion channels. The new PAC and LAPD variants, generated throughout this thesis, generally inform the engineering of synthetic R/FR-sensitive photoreceptors. Intermolecular interactions between sensor and effector modules play important functional roles but also complicate receptor engineering. This work identifies the homologous exchange of sensor and effector modules as a valid strategy for the creation of novel R/FR-sensitive photoreceptors.