Studies of light-induced structural changes in phytochrome using spin-label electron-electron double resonance
Final Report Abstract
A structural model derived from the low-resolution crystal structure of Agpl and a histidine kinase of Thermotoga maritima predicts a parallel arrangement of both subunits in the dimer. In crystals of N-terminal PAS/GAF/PHY fragments often the same arrangement is found, and also the histidine kinase subunits are parallel to each other. Antiparallel arrangements have however been described for crystals of N-terminal fragments of cyanobacterial phytochromes Cph1 and Cph2 and also for an N-terminal Agpl fragment. The PELDOR data indicate that in the full-length protein in solution both subunits are parallel to each other. In this respect electron microscope results on Deinococcus radiodurans bacteriophytochrome are confirmed. Detailed PELDOR and cw-EPR measurements of most mutant samples before and after photoconversion yielded so far no significant differences between Pr and Pfr in the full-length phytochrome. This suggests that protein conformational changes that result in modulation of His kinase activity are very small. Recently, protein conformational changes have been reported for the PCM of Deinococcus phytochrome by comparing Pr and Pfr crystal structures and SAXS measurements in solution. These studies were however carried out only with fragments (PAS-GAF-PHY and PAS-GAF ) of the full-length phytochrome lacking the histidine kinase unit. Whether these motions are also possible in the full-length protein is unclear. Therefore, additional PELDOR studies on similar protein fragments of Agpl or Deinococcus phytochrome are indispensable to shed light on the possibly different behavior of full-length protein and protein fragments. With the Deinococcus data available, it is now possible to devise spin-label positions that are ideally suited to track light-induced conformational changes. Temperature effect. During our measurements on His kinase activity of Agpl, we found an interesting effect of the temperature on kinase activity. Further investigations showed that spectra properties are also changed by elevated temperature in an unexpected manner. Phytochromes might also act as temperature sensors. FRET. Labeling Agpl with two different fluorescent dyes allowed FRET measurements in a steady-state device. Light-induced changes are indicative for changes of the local environment at above mentioned positions. Whether FRET changes are indicative for distance changes remains to be determined.
Publications
- (2011) Molecular Studies on Light-induced Protein Conformational Changes on Agrobacterium tumefaciens Phytochrome, Agp1. Ph.D. thesis, KIT Karlsruhe
Njimona, I
- Temperature effects on Agrobacterium phytochrome Agp1. PLoS ONE 2011, 6, e25977
Njimona, I.; Lamparter, T.
(See online at https://doi.org/10.1371/journal.pone.0025977) - Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr. Journal of Biological Chemistry 2013, 288, 31738-3175
Zienicke, B.; Molina, I.; Glenz, R.; Singer, P.; Ehmer, D.; Escobar, F. V.; Hildebrandt, P.; Diller, R.; Lamparter, T.
(See online at https://dx.doi.org/10.1074/jbc.M113.479535)
