Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants

Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues

L'Ecologisme en Allemagne et en France : deux modes différents de construction d'un nouvel acteur politique

SummaryThe N-terminally truncated variant of photoactive yellow protein (Δ25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Δ25-PYP. In the long-lived photocycle intermediate pB, the central β sheet is still intact, as well as a small part of one α helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated

Hydrogen Bonding Controls Excited-State Decay of the Photoactive Yellow Protein Chromophore

International audienceWe have performed excited-state dynamics simulations of a Photoactive Yellow Protein chromophore analogue in water. The results of the simulations demonstrate that in water the chromophore predominantly undergoes single-bond photoisomerization, rather than double-bond photoisomerization. Despite opposite charge distributions in the chromophore, excited-state decay takes place very efficiently from both single- and double-bond twisted minima in water. Radiationless decay is facilitated by ultrafast solvent reorganization, which stabilizes both minima by specific hydrogen bond interactions. Changing the solvent to the slightly more viscous D(2)O leads to an increase of the excited-state lifetime. Together with previous simulations, the present results provide a complete picture of the effect of the protein on the photoisomerization of the chromophore in PYP: the positive guanidinium group of Arg52 favors double-bond isomerization over single-bond isomerization by lowering the barrier for double-bond isomerization, while the hydrogen bonds with Tyr42 and Glu46 enhance deactivation from the double-bond twisted minimum