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Laboratory of Molecular Biophysics
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Previous: Phytochrome light receptors, Up: Switching states, Return to: Contents.
In collaboration with J. Hendriks, L. Hviid, M. van der Horst, K. J. Hellingwerf- University of Amsterdam, The Netherlands, and T. Gensch- Research Centre Jülich, Germany.
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The photoactive yellow protein (PYP) from Halorhodospira halophila is a 14 kDa cytoplasmic photoreceptor protein that has been extensively studied in recent years. The chromophore, responsible for light-activation of PYP, is p-coumaric acid linked to Cys69 via a thiol-ester bond. It undergoes a trans (in the pG ground state) to cis isomerization during the initial stage of the photocycle of PYP (figure 4). After excitation of the ground state, pG, first an early (microsecond), red-shifted intermediate (pR) is formed. Next a long-lived intermediate (pB) with blue-shifted absorption forms that has a life-time of 400 ms before the ground state is reformed (Figure 4).
The longest lived photocycle-intermediate, pB, presumably is the biological signaling state of the photoreceptor, that is likely to bind to a signaling partner. Hence, significant structural rearrangements are expected to distinguish the pB state from the ground state, pG. The experimental findings so far have shown that the conformational changes in the pB state highly depend on the environment of the protein. In particular, ms and ns time-resolved crystallography probing the pB (Genick et al., 1997) and pR state (Perman et al., 1998), as well as X-ray diffraction of a cryo-trapped pre-pR state (Genick et al., 1998), showed very little structural rearrangements outside the chromophore binding pocket. In contrast, thermodynamic studies of PYP in solution indicated large heat capacity changes associated with pB formation and decay, consistent with large structural rearrangements when pB is formed (van Brederode et al., 1996; Hoff et al., 1999). FTIR difference spectroscopy of the amide region also provided evidence for global conformational changes in PYP (Hoff et al., 1999). Furthermore, light-induced hydrogen-deuterium exchange measurements using FTIR-spectroscopy and mass- spectrometry showed that 23% of the amide-groups, that were buried in pG, became solvent-exposed in the pB state (Hoff et al., 1999). An NMR study of PYP in solution indicated that the structure of the pB state was disordered to a significant degree (Craven et al., 2000). The apparent discrepancy between the crystallographic studies and the studies on PYP in solution was resolved when it was shown with FTIR-spectroscopy that the amplitudes of the signals reflecting structural rearrangements in pB-pG difference spectra were strongly decreased in crystalline PYP (Xie et al., 2001).
We used a polarity-sensitive fluorescent probe, Nile Red (NR), to probe the transient exposure of a hydrophobic surface during the photocycle of PYP. Nile Red was found to have no affinity for the pG ground state, whereas binding to the protein in the pB state was observed. From the fluorescence maximum of the pB-associated NR emission a dielectric constant of D = 15-20 is expected for the environment of NR near its binding site, suggesting exposure of an extended hydrophobic surface. Since NR exclusively binds to the pB signaling state of PYP, which is formed only after light- excitation, we were able to describe the binding of Nile Red to pB using a single second order rate constant and characterize the kinetics of this light-induced conformational changes. By laser flash photolysis in combination with transient absorption and fluorescence measurements, both the kinetics of the optical transitions of the photo- receptor and the kinetics of the conformational changes were determined (Figure 4). Both fluorescence rise and fluorescence decay of Nile Red were found to be slower than the optical photocycle transitions, indicating that the structural changes lag behind the changes in conformation and protonation state of the chromophore (Hendriks et al. submitted).
van Brederode, M. E., W. D. Hoff, I. H. Van Stokkum, M. L. Groot, and K. J. Hellingwerf. (1996). Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein. Biophys J. 71, 365-380.
Craven, C. J., N. M. Derix, J. Hendriks, R. Boelens, K. J. Hellingwerf, and R. Kaptein. (2000). Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: Hydrogen- deuterium exchange data and pH studies. Biochemistry 39, 14392-14399
Genick, U. K., G. E. Borgstahl, K. Ng, Z. Ren, C. Pradervand, P. M. Burke, V. Srajer, T. Y. Teng, W. Schildkamp, D. E. McRee, K. Moffat, and E. D. Getzoff. (1997). Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science 275, 1471-1475.
Genick, U. K., S. M. Soltis, P. Kuhn, I. L. Canestrelli, and E. D. Getzoff. (1998) Structure at 0.85 Å resolution of an early protein photocycle intermediate Nature 392, 206-209
Hendriks, J., Gensch, T., Hviid, L., van der Horst, M., Hellingwerf, K. J. and van Thor, J. J. (2001) Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile Red. (Submitted)
Hoff, W. D., A. Xie, I. H. Van Stokkum, X. J. Tang, J. Gural, A. R. Kroon, and K. J. Hellingwerf. (1999). Global conformational changes upon receptor stimulation in photoactive yellow protein. Biochemistry 38, 1009-1017.
Perman B, Srajer V, Ren Z, Teng T, Pradervand C, Ursby T, Bourgeois D, Schotte F, Wulff M, Kort R, Hellingwerf K, Moffat K (1998) Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle. Science 279, 1946-1950
Xie, A., L. Kelemen, J. Hendriks, B. J. White, K. J. Hellingwerf, and W. D. Hoff. (2001). Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation. Biochemistry. 40, 1510-1517
Previous: Phytochrome light receptors, Up: Switching states, Return to: Contents.