Michael A. van der Horst

Initial characterization of the primary photochemistry of AppA, a blue-light-using flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide chromophore to a conserved tyrosine?

Abstract The flavin adenine dinucleotide (FAD)–containing photoreceptor protein AppA (in which the FAD is bound to a novel so-called BLUF domain) from the purple nonsulfur bacterium Rhodobacter sphaeroides was previously shown to be photoactive by the formation of a slightly redshifted long-lived intermediate that is thought to be the signaling state. In this study, we provide further characterization of the primary photochemistry of this photoreceptor protein using UV–Vis and Fourier-transform infrared spectroscopy, pH measurements and site-directed mutagenesis. Available evidence indicates that the FAD chromophore of AppA may be protonated in the receptor state, and that it becomes exposed to solvent in the signaling state. Furthermore, experimental data lead to the suggestion that intramolecular proton transfer (that may involve [anionic] Tyr-17) forms the basis for the stabilization of the signaling state.

Photoisomerization and Photoionization of the Photoactive Yellow Protein Chromophore in Solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.

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.

The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation.

It is shown that the N‐terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N‐terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N‐terminal domain is linked through a hydrogen‐bonding network with the chromophore‐containing domain of the protein. Furthermore, deletion of the N‐terminal domain completely abolishes the non‐linearity of the Arrhenius plot of the rate of ground state recovery.

Initial photo-induced dynamics of the photoactive yellow protein chromophore in solution

Abstract The initial photoinduced dynamics of thiomethyl p -coumaric acid (TMpCA) in solution has been studied with dispersed time-resolved pump–probe spectroscopy with a ∼100 fs instrument response and extending over a wavelength range of ∼300 to ∼600 nm. TMpCA is a model chromophore for the intrinsic chromophore found in photoactive yellow protein (PYP). Stimulated emission from the chromophore is quenched on a timescale similar to chromophore within the PYP protein. A product state absorption is also observed and is formed earlier than the relaxation of the excited state and that of an observed transient intermediate.

Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis

Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with photochemistry based on trans to cis isomerization of its p-coumaric acid (pCA) chromophore. The addition of trans-locked pCA to I. loihiensis increases biofilm formation, whereas cis-locked pCA decreases it. This demonstrates that the PYP homologue regulates biofilm formation in I. loihiensis, revealing an unexpected functional versatility in the PYP family of photoreceptors. These results imply that I. loihiensis thrives not only in the deep sea but also near the water surface and provide an example of genome-based discovery of photophysiological responses. The use of locked pCA analogs is a novel and generally applicable pharmacochemical tool to study the in vivo role of PYPs irrespective of genetic accessibility. Heterologously produced PYP from I. loihiensis (Il PYP) absorbs maximally at 446 nm and has a pCA pK(a) of 3.4. Photoexcitation triggers the formation of a pB signaling state that decays with a time constant of 0.3 s. FTIR difference signals at 1726 and 1497 cm(-1) reveal that active-site proton transfer during the photocycle is conserved in Il PYP. It has been proposed that a correlation exists between the lifetime of a photoreceptor signaling state and the time scale of the biological response that it regulates. The data presented here provide an example of a protein with a rapid photocycle that regulates a slow biological response.

Photoreceptor proteins from purple bacteria

Purple bacteria contain representatives of four of the six main families of photoreceptor proteins: phytochromes, BLUF domain containing proteins, xanthopsins (i.e., photoactive yellow proteins), and phototropins (containing one or more light, oxygen, or voltage (LOV) domains). Most of them have a function in adjusting the cellular transcript profile to the ambient light climate. Here we will discuss, with examples of the most important representative(s) of each of these four families, the interdependent topics: (i) the proteins’ biological functions and their molecular context, (ii) the results of ultra-fast and static spectroscopic studies, and (iii) structural alterations required for initiation of signal transfer, as resolved with transient spectroscopy and the methodology of structural biology.

Measuring and modelling dynamical changes in the structure of photoactive yellow protein

Biological photoreceptors are very suitable for studies of the structure–function relationship in proteins. Of the many possible candidates, from at least six different families, we discuss here photoactive yellow protein (PYP, a member of the Xanthopsin family). The excellent physical and photochemical stability of PYP has allowed recording of a large amount of data relevant for this characteristic. The timescales relevant for functional transitions in PYP are most easily studied with transient spectroscopy. Application of the combination of UV/Vis and FTIR spectroscopy has revealed (besides multiple electronically excited states) the subsequent involvement of the following transient intermediates: I0, pR1, pR2, pB′, pB and pBdeprot. The structures of these transient intermediates have been characterized with general structural techniques like X-ray diffraction and NMR spectroscopy. Most extensive results have been obtained with X-ray diffraction, applied both in a time-resolved mode and by making use of low-temperature trapping. This has led to a detailed description of the reversible spatial changes in PYP that follow light activation. The structure of the transient intermediates, however, is exquisitely sensitive to constraints from the molecular environment of the protein, like the absence/presence of a crystalline lattice. Extrapolating these results to the signalling state of PYP in vivo is extremely complicated but also very challenging. Rather than measure, one can also simulate and/or calculate the spatial structures of transient intermediates of PYP. This challenge is most straightforwardly tackled with a detailed analysis of the intrinsic dynamics of the stable receptor state of PYP. Such studies have revealed striking similarities in the dynamics of all PAS domain proteins of which a spatial structure has been determined. The next challenge is to properly describe the change in configuration of the chromophore, induced by light, and to expand such simulations to the timescale relevant for completion of this photocycle.