Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect 13C NMR spectroscopy

Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated ¹³C_γ resonances in sensitive and well-resolved two-dimensional ¹³C-¹H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK_a values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41–11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.     

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.     

The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK(a) of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK(a) regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK(a) of the chromophore. R52 is found to flip out during the formation of PYP(M). The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK(a) regulation during the photocycle.     

Early molecular events in the photoactive yellow protein: role of the chromophore photophysics.

We report a comparative study of the isomerization reaction in native and denatured photoactive yellow protein (PYP) and in various chromophore analogues in their trans deprotonated form. The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy. The free p-hydroxycinnamate (pCA(2-)) and its amide analogue (pCM(-)) are found to display a quite different transient spectroscopy from that of PYP. The excited-state deactivation leads to the formation of the ground-state cis isomer without any detectable intermediate with a mechanism comparable to trans-stilbene photoisomerization. On the contrary, the early stage of the excited-state deactivation of the free thiophenyl-p-hydroxycinnamate (pCT(-)) and of the denatured PYP is similar to that of the native protein. It involves the formation of an intermediate absorbing in the spectral region located between the bleaching and gain bands in less than 2 ps. However, in these two cases, the formation of the cis isomer has not been proved yet. This difference with pCA(-) and pCM(-) might result from the fact that, in the thioester substituted chromophore, simultaneous population of two quasi-degenerate excited states occurs upon excitation. This comparative study highlights the determining role of the chromophore structure and of its intrinsic properties in the primary molecular events in native PYP.     

Ultrafast light-induced response of photoactive yellow protein chromophore analogues.

The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.     

Exploring chromophore--protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy

Autofluorescent proteins of the GFP family all share the same three-dimensional beta-can fold; yet they exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic characterization of the green fluorescent protein cmFP512 from Cerianthus membranaceus, a nonbioluminescent, azooxanthellate cnidarian, which has only approximately 22% sequence identity with Aequorea victoria GFP. The X-ray structure, obtained by molecular replacement at a resolution of 1. 35 A, shows the chromophore, formed from the tripeptide Gln-Tyr-Gly, in a hydrogen-bonded cage in the center of an 11-stranded beta-barrel, tightly restrained by adjacent residues and structural water molecules. It exists in a neutral (A) and an anionic (B) species, with absorption/emission maxima at 392/460 (pH 5) and 503/512 nm (pH 7). Their fractional populations and peak positions depend sensitively on pH, reflecting protonation of groups adjacent to the chromophore. The pH dependence of the spectra is explained by a protonation mechanism involving a hydrogen-bonded cluster of charged/polar groups. Cryospectroscopy at 12 K was also performed to analyze the vibronic coupling of the electronic transitions.     

Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.     

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.     

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.     

Signal transduction in the photoactive yellow protein. I. Photon absorption and the isomerization of the chromophore

Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. The calculations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization transition state. Although signaling from PYP is thought to involve partial unfolding of the protein, the mechanical effects accompanying isomerization do not appear to directly destabilize the protein.     

Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.     

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.     

Water structural changes involved in the activation process of photoactive yellow protein

Fourier transform infrared (FTIR) spectroscopy was applied to the blue-light photoreceptor photoactive yellow protein (PYP) to investigate water structural changes possibly involved in the photocycle of PYP. Photointermediates were stabilized at low temperature, and difference IR spectra were obtained between intermediate states and the original state of PYP (pG). Water structural changes were never observed in the >3570 cm(-)(1) region for the intermediates stabilized at 77-250 K, such as the red-shifted pR and blue-shifted pB intermediates. In contrast, a negative band was observed at 3658 cm(-)(1) in the pB minus pG spectrum at 295 K, which shifts to 3648 cm(-)(1) upon hydration with H(2)(18)O. The high frequency of the O-H stretch of water indicates that the water O-H group does not form hydrogen bonds in pG, and newly forms these upon pB formation at 295 K, but not at 250 K. Among 92 water molecules in the crystal structure of PYP, only 1 water molecule, water-200, is present in a hydrophobic core inside the protein. The amide N-H of Gly-7 and the imidazole nitrogen atom of His-108 are its possible hydrogen-bonding partners, indicating that one O-H group of water-200 is free to form an additional hydrogen bond. The water band at 3658 cm(-)(1) was indeed diminished in the H108F protein, which strongly suggests that the water band originates from water-200. Structural changes of amide bands in pB were much greater in the wild-type protein at 295 K than at 250 K or in the H108F protein at 295 K. The position of water-200 is >15 A remote from the chromophore. Virtually no structural changes were reported for regions larger than a few angstroms away from the chromophore, in the time-resolved X-ray crystallography experiments on pB. On the basis of the present results, as well as other spectroscopic observations, we conclude that water-200 (buried in a hydrophobic core in pG) is exposed to the aqueous phase upon formation of pB in solution. In neither crystalline PYP nor at low temperature is this structural transition observed, presumably because of the restrictions on global structural changes in the protein under these conditions.     

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.     

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in 相似文献   

Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: a pH-driven color transition.

Photoactive yellow protein (PYP) is a blue light sensor present in the purple photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) of 446 nm. The anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen-bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I(2)) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a key factor in controlling the color and photocycle kinetics of PYP is this H-bonding system. To further investigate this, we have now characterized Glu46Asp and Glu46Ala mutants. The ground-state absorption spectrum of the Glu46Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species: a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-shifted deprotonated (basic) form (lambda(max) = 444 nm). Both of these species are photoactive. A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive red-shifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm). We attribute these spectral transitions to protonation/deprotonation of the phenolate oxygen of the chromophore. This is demonstrated by FT Raman spectra. Dark recovery kinetics (return to the unphotolyzed state) were found to vary appreciably between these various photoactive species. These spectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.     

pH dependence of the photoactive yellow protein photocycle investigated by time-resolved crystallography

Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values.     

On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

Prior experimental observations, as well as theoretical considerations, have led to the proposal that C₄-C₇ single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C₄-C₇ single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C₄-C₇ single-bond rotation in PYP is not an alternative to C₇=C₈ double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore.     

Molecular dynamics study of femtosecond events in photoactive yellow protein after photoexcitation of the chromophore

Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues. Proteins 32:268–275, 1998. © 1998 Wiley-Liss, Inc.     

Molecular dynamics simulations of photoactive yellow protein (PYP) in three states of its photocycle: a comparison with X-ray and NMR data and analysis of the effects of Glu46 deprotonation and mutation

Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known. Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln) and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data, and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant. Electronic supplementary material to this article is available at and is accessible for athorized users. Electronic Publication