Computergestuetztes design von kuenstlichen Protein-Photorezeptoren fuer optogenetische Anwendungen
Biochemie
Zusammenfassung der Projektergebnisse
A total of 5 projects were worked on that all had implications regarding the design of photoactivatable proteins/peptides and/or computational modeling of photoreceptors. The most important outcome of the project is the development of light- and, conincidentally, temperature sensitive Cas9 variants, by creating chimeric proteins between Cas9 and RsLOV, a recently discovered photoreceptor protein. Cas9, recently discovered in the context of CRISPR (Clustered-Regularly-Interspaced-Short-Palindromic-Repeats) bacterial adapative immunity, is a DNA-endonuclease that uses an RNA molecule (gRNA) to determine which DNA sequence to cut. Cas9 has enabled genome engineering and transcriptional modulation across many model organisms and cell lines. Aided by computational modeling, we created a large domain insertion library of Cas9 of RsLOV. Screening this library for switchable constructs using an E.coli based high-throughput assay in combination with fluorescent-activated cell sorting (FACS) yielded one moderately lightswitchable (paRC9) and, unexpectedly, one strongly temperature-switchable (between 29 and 37°C) variant (tsRC9). This work is of relevance in two areas: first, (de-)activatable Cas9 variants are potentially useful for several reasons, namely spatiotemporal control over activities of interest, as well as reduction of Cas9 off-target effects and post-cleavage DNA residence times. Second, a general approach to create light-switchable variants of arbitrary effectors of interest (particularly monomeric) remains an unsolved problem in photoreceptor engineering and optogenetics. Investigating novel photoreceptors in this regard can thus contribute to the toolbox available for photoreceptor engineering. The surprising finding that RsLOV can render Cas9 temperature-sensitive is also remarkable, as it represents the first report of a photoreceptor bestowing temperature-sensitivity onto an effector and also the first report of a directly temperature-sensitive Cas9. As an initial application of this, we present temperature-controlled repression of arbitrary target genes in E.coli.. A second relevant outcome was the design of an enzyme that should enable recombinant production of proteins with adhesive proteins mediated by unnatural amino acids. We used computation to redesign an aminoacyl-tRNA synthetase (aaRS) active site towards recognizing photocaged variants of tyrosine and the adhesive unnatural amino acid Dopa. Mutations at 26 sites and a corresponding sequence space of ~1034 were considered in silico, and a focused library of size 108 with sensible diversity at 17 positions was generated based on computational models. High-activity aaRS for both ONBY and ONB-Dopa were isolated from this library using established screening methods. ONB-DopaRS enabled the, to our knowledge, first demonstration of recombinant multi-site incorporation of a Dopa analog into proteins by means of an engineered aaRS. ONBYRS had higher activity than other synthetases for ONBY that were engineered using traditional methods. The obtained results thus represent significant progress both in the general field of aaRS engineering as well as in the recombinant production of proteins with adhesive potential. Further outcomes obtained during the course of the project were the development of an apparatus that allowed spectroscopic investigation of light-sensitive proteins at highthroughput, which is a necessary capability for engineering photoreceptors. Additionally, a method was developed to allow for efficiently constructing DNA libraries for a certain class of photoreceptor-effector fusions, and lastly, computational modeling was used to interpret spectroscopically detected conformational changes in a photoreceptor upon light-exposure.
Projektbezogene Publikationen (Auswahl)
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Light-Induced Rearrangement of the β5 Strand in the BLUF Photoreceptor SyPixD (Slr1694). J Phys Chem Lett 6, 4749–4753 (2015)
Mehlhorn, J, Lindtner, T, Richter F, et al.
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Upgrading a microplate reader for photobiology and all-optical experiments. Photochem. Photobiol. Sci. 14, 270–279 (2015)
Richter, F, Scheib US, Mehlhorn J, Schubert R, Wietek J, et al.
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Library-Aided Probing of Linker Determinants in Hybrid Photoreceptors. ACS Synth Biol (2016)
Ohlendorf, R, Schumacher, C. H., Richter, F. & Möglich, A.
