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Phytochrome from photochemistry to signalling: MAS NMR studies of Cph1 structure/function at atomic resolution

Subject Area Biophysics
Analytical Chemistry
Structural Biology
Term from 2019 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 417685888
 
Final Report Year 2024

Final Report Abstract

Phytochromes are photoreceptor molecules used by all plants and many bacteria to sense and respond to their light environment, characteristically using a bilin co-factor to absorb photons, and thereby shifting their absorption wavelength (typically from the red to the far-red region, thereby the Pr and Pfr states) and de/activating their signal to the cell. We aimed to use MAS (magic-angle spinning nuclear) NMR to study how these and other characters connect to changes in molecular structure in model prokaryotic phytochromes such as Cph1, Cph2-like phytochromes and various cyanobacteriochromes (CBCRs). We were also able to develop new techniques that might prove generally useful in MAS NMR and other methods. We note that our work was drastically restricted by the Covid pandemic 03.2020–12.2021. A major collaborative project focused on the Y176H mutant of Cph1 that is not only strongly fluorescent but in plant phytochromes signals to the cell even in total darkness. We were able to solve the crystal structure of the mutant, revealing that although the chromophore was in the Pr-typical conformation, the protein showed characteristics typical of Pfr. We studied this remarkable uncoupling of chromophore and protein extensively, identifying a very long-lived excited state that is unable to make the transition towards Pfr as the origin of the strong fluorescence. We also wanted to study the intramolecular signal connecting the chromophore to a portion of the molecule – the tongue – known to undergo radical refolding upon light activation. This work was carried out on wild-type Cph1 in collaboration with the Oschkinat group in Berlin, allowing DNP enhancement of the MAS NMR signal to be exploited. Numerous technical problems were encountered such that even now only part of the planned program has been completed. We were additionally surprised to find that the expected signals related to the Pfr but not the Pr state could be resolved. We studied ultrafast changes in Cph1 during Pfr back-conversion to Pr too, concluding that dynamics begin with chromophore isomerization within 1 ps, possibly as a result of electrostatic forces. CBCR-GAF domains are small photochromic biliproteins that exhibit remarkable variation in absorption wavelength. We focused on one type in which a red-absorbing dark state similar to Pr of canonical phytochromes forms a hypsochromically shifted green-absorbing photoproduct. MAS NMR and QM/MM calculations provided insight into changes in and around the chromophore and the role of a conserved tyrosine in spectral tuning. Separately, we showed that this tyrosine is important in Pr→Pfr photoconversion, providing a basis for rational design of optimized phytochromes in biotechnological applications. Photoproduct thermal reversion is a problem in studying short-lived photoproducts. Thus we developed an innovative approach for photoproduct stabilization by incorporating the protein into trehalose glasses (TGs). We showed that the TGs inhibit thermal reversion of the incorporated proteins for weeks at room temperature with preserving structural and functional integrity of the proteins. Even with DNP enhancement, MAS NMR is an insensitive method, thereby requiring large sample amounts, whereas the samples themselves must be spun in rotors at extremely high speeds, restricting the possible size of the rotor. Thus, we evolved ultracentrifugation techniques that allow proteins to be concentrated into the sample rotor.

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