Biomedical subjects

Michael A van der Horst

Publications and source records attributed to Michael A van der Horst.

10 recordsLinked to original sources

PubMed2005

Controlled reduction of the humidity induces a shortcut recovery reaction in the photocycle of photoactive yellow protein.

The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.

Bacterial Proteins↗

PubMed2005

Ultrafast dynamics of isolated model photoactive yellow protein chromophores: "Chemical perturbation theory" in the laboratory.

Pump-probe and pump-dump probe experiments have been performed on several isolated model chromophores of the photoactive yellow protein (PYP). The observed transient absorption spectra are discussed in terms of the spectral signatures ascribed to solvation, excited-state twisting, and vibrational relaxation. It is observed that the protonation state has a profound effect on the excited-state lifetime of p-coumaric acid. Pigments with ester groups on the coumaryl tail end and charged phenolic moieties show dynamics that are significantly different from those of other pigments. Here, an unrelaxed ground-state intermediate could be observed in pump-probe signals. A similar intermediate could be identified in the sinapinic acid and in isomerization-locked chromophores by means of pump-dump probe spectroscopy; however, in these compounds it is less pronounced and could be due to ground-state solvation and/or vibrational relaxation. Because of strong protonation-state dependencies and the effect of electron donor groups, it is argued that charge redistribution upon excitation determines the twisting reaction pathway, possibly through interaction with the environment. It is suggested that the same pathway may be responsible for the initiation of the photocycle in native PYP.

Bacterial Proteins↗

PubMed2005

The solution structure of a transient photoreceptor intermediate: Delta25 photoactive yellow protein.

The N-terminally truncated variant of photoactive yellow protein (Delta25-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 Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha 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.

Bacterial Proteins↗

PubMed2004

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.

Coumaric Acids↗

PubMed2004

Photoreceptor proteins, "star actors of modern times": a review of the functional dynamics in the structure of representative members of six different photoreceptor families.

Six well-characterized photoreceptor families function in Nature to mediate light-induced signal transduction: the rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. The first three catalyze E/Z isomerization of retinal, phytochromobilin, and p-coumaric acid, respectively, while the last three all have a different flavin-based photochemistry. For many of these photoreceptor proteins, (many of) the details of the conversion of the light-induced change in configuration of their chromophore into a signaling state and eventually a biological response have been resolved. Some members of the rhodopsins, the xanthopsins, and the phototropins are so well characterized that they function as model systems to study (receptor) protein dynamics and (un)folding.

Animals↗

PubMed2004

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments.

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing "locked" chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.

Bacterial Proteins↗

PubMed2003

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate.

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.

Amino Acid Substitution↗

PubMed2003

Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy.

Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.

Amino Acids↗

PubMed2003

PAS domains. Common structure and common flexibility.

PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved alpha/beta-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.

Amino Acid Motifs↗

PubMed2003

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?

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.

Amino Acid Sequence↗