|
Laboratory of Molecular Biophysics
|
Previous: Green Fluorescent Protein, Next: Photoactive yellow protein , Up: Switching states, Return to: Contents.
in collaboration with B. Borucki, H. Otto , T. Lamparter , J. Hughes, M. P. Heyn -Freie Universität Berlin, Germany- and K. J. Hellingwerf and W. Crielaard - University of Amsterdam, the Netherlands.
|
From the Synechocystis PCC 6803 genome sequencing project, a phytochrome homologue was identified that upon heterologous expression in Escherichia coli yielded apo-phytochrome that was reconstituted with linear tetrapyrrole chromophores to produce spectrally active holo-phytochrome (Hughes et al., 1997). This light-receptor, Cph1, is a light-regulated histidine-kinase of the two-component type, that shows red/far-red light induced optical transitions between the 'Pfr' (absorbs in the 'far-red'; 704 nm) and 'Pr' (absorbs in the 'red'; 655 nm) states, typical of phytochromes. Phototransformation from Pr to Pfr is known to involve a Z,E photoisomerisation around the 15, 16-double bond connecting the C- and D-rings of the linear tetrapyrrole (Figure 3).
Time-resolved absorption measurements after laser flash photolysis of the Pr state of Cph1 provided the kinetics of the transient formation of spectral intermediates between the Pr and the Pfr states, in addition to the associated difference spectra. 5 relevant time-constants were isolated from the time-resolved measurements, ranging from the ms to s time-domain. The kinetics of proton release - and proton uptake reactions during the Pr- to Pfr transition were determined with time-resolved pH-indicator experiments. After excitation of the Pr state at neutral pH approximately one proton was released (with time- constants of ~4 and ~40 ms) but also later partially taken back up again (~270 ms), synchronous with the formation of the Pfr state. The kinetics of proton release and uptake fitted with a selected set of rate-constants isolated from the photo-cycle measurements. The protonation state of the chromophores, in particular at the position of the C-ring nitrogen, in Pr and Pfr, has not been determined conclusively using FT-Raman and FT-IR spectroscopy. Analysis of the pH-dependence of the Pr absorption spectra and the calculated UV/Vis absorption spectra of Pfr from 6.5 to 9.0 and comparison with model compound studies suggested that the phycocyanobilin chromophore deprotonates at the C-ring nitrogen at alkaline pH in both Pr and Pfr with an approximate pKA of 9.5. The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pKA changes in the protein moiety, and not of the chromophore, which are linked to conformational transitions occurring around 4 ms and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome (van Thor et al., 2001).
Jasper van Thor and Louise N. Johnson
A phytochrome-type of photoreceptor, called Ppr, was discovered in the purple bacterium R. centenum (Jiang et al., 1999). This photoreceptor protein can be regarded as a fusion protein consisting of a Photoactive Yellow Protein (PYP) domain and a phytochrome domain. The N-terminal domain has high sequence homology with PYP's, whereas the C-terminal domain has high sequence homology with cyanobacterial and also plant type of phytochromes. In addition, functional homology of the N-terminal 'PYP domain' of Ppr with PYP's could also be demonstrated with respect to its spectroscopic properties and photochromic behaviour (Jiang et al., 1999). The C-terminal domain shows sequence homology to phytochromes and other bilin lyases, but so far has not been reported to bind phycocyanin or phytochromobilin. In the case of Ppr the structure of the apo- phytochrome domain may represent the biologically active form if the N-terminal PYP domain is the only active sensor-domain in the receptor.
Hughes J, Lamparter T, Mittmann F, Hartmann E, Gartner W, Wilde A, Borner T (1997) A prokaryotic phytochrome. Nature 386, 663
Jiang Z, Swem LR, Rushing BG, Devanathan S, Tollin G, Bauer CE (1999) Bacterial photoreceptor with similarity to photoactive yellow protein and plant phytochromes. Science 285, 406-409
Jasper J. van Thor, Berthold Borucki, Wim Crielaard, Harald Otto, Tilman Lamparter, Jon Hughes, Klaas J. Hellingwerf, and Maarten P. Heyn. (2001) Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1. Biochemistry 40, 11460-11471
Previous: Green Fluorescent Protein, Next: Photoactive yellow protein , Up: Switching states, Return to: Contents.