Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy
Kurttila, M., Stucki-Buchli, B., Rumfeldt, J., Schroeder, L., Häkkänen, H., Liukkonen, A., Takala, H., Kottke, T., & Ihalainen, J. (2021). Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy. Physical Chemistry Chemical Physics, 23(9), 5615-5628. https://doi.org/10.1039/d0cp06553f
Published inPhysical Chemistry Chemical Physics
© the Owner Societies 2021
Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels. ...
PublisherRoyal Society of Chemistry (RSC)
Publication in research information system
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Related funder(s)Jane and Aatos Erkko Foundation; Academy of Finland
Funding program(s)Foundation; Academy Project, AoF; Research post as Academy Research Fellow, AoF
Additional information about fundingThe work of BS-B has been supported by a grant from the Swiss National Science Foundation (P2ZHP2_164991). JAI acknowledges the Academy of Finland (296135 and 332742) and the Jane and Aatos Erkko foundation. HT acknowledges the Academy of Finland (285461 and 330678). LS acknowledges a fellowship of the Studienstiftung des Deutschen Volkes. TK acknowledges a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (KO3580/4-2). ...
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