Johnny Hendriks

Transient Exposure of Hydrophobic Surface in the Photoactive Yellow Protein Monitored with Nile Red

In this study we have investigated binding of the fluorescent hydrophobicity probe Nile Red to the photoactive yellow protein (PYP), to characterize the exposure and accessibility of hydrophobic surface upon formation of the signaling state of this photoreceptor protein. Binding of Nile Red, reflected by a large blue shift and increase in fluorescence quantum yield of the Nile Red emission, is observed exclusively when PYP resides in its signaling state. N-terminal truncation of the protein allows assignment of the region surrounding the chromophore as the site where Nile Red binds to PYP. We also observed a pH dependence of the affinity of Nile Red for pB, which we propose is caused by pH dependent differences of the structure of the signaling state. From a comparative analysis of the kinetics of Nile Red binding and transient absorption changes in the visible region we can conclude that protonation of the chromophore precedes the exposure of a hydrophobic surface near the chromophore binding site, upon formation of the signaling state. Furthermore, the data presented here favor the view that the signaling state is structurally heterogeneous.

On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.

Protonation/Deprotonation Reactions Triggered by Photoactivation of Photoactive Yellow Protein from Ectothiorhodospira halophila

Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.

The primary photoreaction of photoactive yellow protein (PYP): anisotropy changes and excitation wavelength dependence

Abstract The absorption and stimulated emission changes in the first 535 ps of the PYP photocycle can be described by four life times of 0.7, 6.3, and 220 ps and long lived. Two intermediates, I0 and I 0 ‡ , were identified. We did not obtain indications for a significant excitation wavelength dependent primary photochemistry as found in low temperature absorption spectroscopy. The anisotropy of the primary photoproduct I0 and its successor I 0 ‡ amounts to 0.3 – significantly lower than that of the bleached ground state (0.4). This distinctive change of the transition dipole moment orientation in the product state (24°) reflects changes of the chromophore geometry and electron density distribution caused by the photoisomerisation.

Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila

We have studied the kinetics of the blue light‐induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time‐resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue‐shifted signaling state pB, the light‐induced branching reaction showed a 1000‐fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue‐shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate‐controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis→trans isomerization of the 4‐hydroxy‐cinnamyl chromophore of PYP in its pB state.

PH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore

The recovery reaction of the signaling state of photoactive yellow protein includes the following: (i) deprotonation of the p-coumaryl chromophore, (ii) refolding of the protein, and (iii) chromophore re-isomerization from the cis to the trans configuration. Through analysis of the pH dependence of this recovery reaction, we were able to provide proof for the existence of an additional photocycle intermediate. The spectral similarity between this new intermediate and the dark state indicates that the new intermediate has a deprotonated chromophore, which may facilitate chromophore re-isomerization. This spectral similarity also explains why this new intermediate has not been noticed in earlier studies. For our data analysis we introduce a photocycle model that takes into account the effect of the specific light regime selected, a model that was also used for simulations.

On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein

The redox‐midpoint potential of the FAD chromophore in the BLUF domain of anti‐transcriptional regulator AppA from Rhodobacter sphaeroides equals ∼−260 mV relative to the calomel electrode. Altering the structure of its chromophore‐binding pocket through site‐directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox‐midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which may rationalize the primary photochemistry observed in these three flavin‐containing photoreceptor families. These results also imply that LOV domains, among the flavin‐containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark.

On the configurational and conformational changes in Photoactive Yellow Protein that leads to signal generation in Ectothiorhodospira halophila

Photoactive Yellow Protein (PYP), a phototaxis photoreceptor from Ectothiorhodospira halophila, is a small water-soluble protein that iscrystallisable and excellently photo-stable. It can be activated with light(λmax= 446 nm), to enter a series of transientintermediates that jointly form the photocycle of this photosensor protein.The most stable of these transient states is the signalling state forphototaxis, pB.The spatial structure of the ground state of PYP, pG and the spectralproperties of the photocycle intermediates have been very well resolved.Owing to its excellent chemical- and photochemical stability, also the spatialstructure of its photocycle intermediates has been characterised with X-raydiffraction and multinuclear NMR spectroscopy. Surprisingly, the resultsobtained showed that their structure is dependent on the molecular contextin which they are formed. Therefore, a large range of diffraction-,scattering- and spectroscopic techniques is now being employed to resolvein detail the dynamical changes of the structure of PYP while it progressesthrough its photocycle. This approach has led to considerable progress,although some techniques still result in mutually inconsistent conclusionsregarding aspects of the structure of particular intermediates.Recently, significant progress has also been made with simulations withmolecular dynamics analyses of the initial events that occur in PYP uponphoto activation. The great challenge in this field is to eventually obtainagreement between predicted dynamical alterations in PYP structure, asobtained with the MD approach and the actually measured dynamicalchanges in its structure as evolving during photocycle progression.

Isolation, reconstitution and functional characterisation of the Rhodobacter sphaeroides photoactive yellow protein.

We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned. Reconstitution of the his‐tagged purified apo‐protein with 4‐hydroxy‐cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm. This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy. Moreover, reconstitution with the ‘locked’ analogue 7‐hydroxy‐coumarin‐3‐carboxylic acid yields only a single absorbance peak at 441 nm. The 446 nm and 360 nm species are part of a temperature‐ and pH‐dependent equilibrium. Photoactivation of the protein leads to formation of a blue‐shifted intermediate as in other PYPs, with a 100‐fold increased groundstate recovery rate (k pB→pG=500 s−1) compared to E‐PYP.

Modeling the functioning of YtvA in the general stress response in Bacillus subtilis

The blue-light photoreceptor YtvA activates the general stress response (GSR) of Bacillus subtilis by activating a large protein complex (the stressosome). We have constructed a model for the YtvAs photocycle, and derived an equation for the fraction of YtvA in the light-induced signaling state at a given light intensity. The model was verified experimentally in vitro on wild type YtvA and on an R63K mutant with faster recovery kinetics. Application of the model to the activation of the GSR at various light intensities in vivo revealed that the GSR is more sensitive to light than would be expected based on YtvAs in vitro kinetics. These results were confirmed with the R63K mutant and a slower-recovering V28I mutant. Additionally, we have demonstrated the presence of a near-UV-light-induced branching reaction that converts the signaling state of YtvA to the dark state. Extension of the model with this reaction shows that it does not contribute significantly to the in vivo blue-light response. The model represents an important step towards a complete systems biology model of the GSR.