Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy
| Autor: | Heikki Häkkänen, Brigitte Stucki-Buchli, Heikki Takala, Lea Schroeder, Janne A. Ihalainen, Tilman Kottke, Alli Liukkonen, Jessica Rumfeldt, Moona Kurttila |
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| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Azides Protein Conformation Phenylalanine spektroskopia Tongue region General Physics and Astronomy fotobiologia 010402 general chemistry Tracking (particle physics) 01 natural sciences 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Spectroscopy Fourier Transform Infrared Amino Acid Sequence Amino Acids Physical and Theoretical Chemistry Fourier transform infrared spectroscopy Bilin 030304 developmental biology 0303 health sciences Binding Sites Staining and Labeling biology Phytochrome Chemistry Deinococcus radiodurans Chromophore Photochemical Processes biology.organism_classification 0104 chemical sciences Kinetics Biophysics proteiinit valokemia Signal transduction Protein Binding Signal Transduction |
| Zdroj: | Physical Chemistry Chemical Physics. 23:5615-5628 |
| ISSN: | 1463-9084 1463-9076 |
| Popis: | 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. peerReviewed |
| Databáze: | OpenAIRE |
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