State Of Photoactive Yellow Protein Research Articles

Length and Charge of the N-terminus Regulate the Lifetime of the Signaling State of Photoactive Yellow Protein.

Photoactive yellow protein (PYP) is one of the most extensively studied photoreceptors. Nevertheless, the role of the N-terminus in the photocycle and structural transitions is still elusive. Here, we attached additional amino acids to the N-terminus of PYP and investigated the effect of the length and charge of additional N-terminal residues using circular dichroism, two-dimensional nuclear magnetic resonance (2D-NMR), transient absorption (TA), and transient grating (TG) spectroscopic techniques. TA experiments showed that, except for negatively charged residues (5D-PYP), additional N-terminal residues of PYP generally enable faster dark recovery from the putative signaling state (pB2) to the ground state (pG). TG data showed that although the degree of structural changes can be controlled by adjusting specific amino acid residues in the extended N-terminus of N-terminal extended PYPs (NE-PYPs), the dark recovery times of wt-PYP and NE-PYPs, except for 5D-PYP, are independent of the structural differences between pG and pB2 states. These results demonstrate that the recovery time and the degree of structural change can be regulated by controlling the length and sequence of N-terminal residues of PYP. The findings in this study emphasize the need for careful attention to the remaining amino acid residues when designing recombinant proteins for genetic engineering purposes.

Absorption wavelength along chromophore low-barrier hydrogen bonds

SummaryIn low-barrier hydrogen bonds (H-bonds), the pKa values for the H-bond donor and acceptor moieties are nearly equal, whereas the redox potential values depend on the H+ position. Spectroscopic details of low-barrier H-bonds remain unclear. Here, we report the absorption wavelength along low-barrier H-bonds in protein environments, using a quantum mechanical/molecular mechanical approach. Low-barrier H-bonds form between Glu46 and p-coumaric acid (pCA) in the intermediate pRCW state of photoactive yellow protein and between Asp116 and the retinal Schiff base in the intermediate M-state of the sodium-pumping rhodopsin KR2. The H+ displacement of only ∼0.4 Å, which does not easily occur without low-barrier H-bonds, is responsible for the ∼50 nm-shift in the absorption wavelength. This may be a basis of how photoreceptor proteins have evolved to proceed photocycles using abundant protons.

Protonation Equilibrium in the Active Site of the Photoactive Yellow Protein.

The role and existence of low-barrier hydrogen bonds (LBHBs) in enzymatic and protein activity has been largely debated. An interesting case is that of the photoactive yellow protein (PYP). In this protein, two short HBs adjacent to the chromophore, p-coumaric acid (pCA), have been identified by X-ray and neutron diffraction experiments. However, there is a lack of agreement on the chemical nature of these H-bond interactions. Additionally, no consensus has been reached on the presence of LBHBs in the active site of the protein, despite various experimental and theoretical studies having been carried out to investigate this issue. In this work, we perform a computational study that combines classical and density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) simulations to shed light onto this controversy. Furthermore, we aim to deepen our understanding of the chemical nature and dynamics of the protons involved in the two short hydrogen bonds that, in the dark state of PYP, connect pCA with the two binding pocket residues (E46 and Y42). Our results support the existence of a strong LBHB between pCA and E46, with the H fully delocalized and shared between both the carboxylic oxygen of E46 and the phenolic oxygen of pCA. Additionally, our findings suggest that the pCA interaction with Y42 can be suitably described as a typical short ionic H-bond of moderate strength that is fully localized on the phenolic oxygen of Y42.

Absorption Wavelength Along Chromophore Low-Barrier Hydrogen Bonds

In low-barrier hydrogen bonds (H-bonds), the pKa values for the H-bond donor and acceptor moieties are nearly equal, whereas the redox potential values depend on the H+ position. Spectroscopic details of low-barrier H-bonds remain unclear. Here, we report the absorption wavelength along low-barrier H-bonds in protein environments, using a quantum mechanical/molecular mechanical approach. Low-barrier H-bonds form-between Glu46 and p-coumaric acid (pCA) in the intermediate pRCW state of photoactive yellow protein and between Asp116 and the Schiff base in the intermediate M-state of the sodium-pumping rhodopsin KR2. The absorption wavelength remains constant at ~460 nm until H+ arrives at the pCA moiety. As H+ moves ~0.4 A and arrives at the pCA moiety, the absorption wavelength is discontinuously shortened to ~420 nm. The step-function-like switching mechanism may be advantageous in the adoption of the specific absorption wavelength of the signaling state prior to time-consuming large structural changes during the photoreceptor photocycle. This may be a basis of how photoreceptor proteins have evolved to proceed photocycles using abundant protons.

Vibrational energy flow in photoactive yellow protein revealed by infrared pump-visible probe spectroscopy.

Vibrational energy flow in the electronic ground state of photoactive yellow protein (PYP) is studied by ultrafast infrared (IR) pump-visible probe spectroscopy. Vibrational modes of the chromophore and the surrounding protein are excited with a femtosecond IR pump pulse, and the subsequent vibrational dynamics in the chromophore are selectively probed with a visible probe pulse through changes in the absorption spectrum of the chromophore. We thus obtain the vibrational energy flow with four characteristic time constants. The vibrational excitation with an IR pulse at 1340, 1420, 1500, or 1670 cm(-1) results in ultrafast intramolecular vibrational redistribution (IVR) with a time constant of 0.2 ps. The vibrational modes excited through the IVR process relax to the initial ground state with a time constant of 6-8 ps in parallel with vibrational cooling with a time constant of 14 ps. In addition, upon excitation with an IR pulse at 1670 cm(-1), we observe the energy flow from the protein backbone to the chromophore that occurs with a time constant of 4.2 ps.

Novel Approach to Excited-State Calculations of Large Molecules Based on Divide-and-Conquer Method: Application to Photoactive Yellow Protein

In this study, the divide-and-conquer (DC) method is extended to configuration-interaction singles, time-dependent density functional, and symmetry-adapted cluster configuration interaction (SACCI) theories for enabling excited-state calculations of large systems. In DC-based excited-state theories, one subsystem is selected as the excitation subsystem and analyzed via excited-state calculations. Test calculations for formaldehyde in water and a conjugated aldehyde demonstrate the high accuracy and effectiveness of these methods. To demonstrate the efficiency of the method, we calculated the π-π* excited state of photoactive yellow protein (PYP). The numerical applications to PYP confirm that the DC-SACCI method significantly accelerates the excited-state calculations while maintaining high accuracy.

Ultrafast Hydrogen-Bonding Dynamics in the Electronic Excited State of Photoactive Yellow Protein Revealed by Femtosecond Stimulated Raman Spectroscopy

The ultrafast structural dynamics in the electronic excited state of photoactive yellow protein (PYP) is studied by femtosecond stimulated Raman spectroscopy. Stimulated Raman spectra in the electronic excited state, S(1), can be obtained by using a Raman pump pulse in resonance with the S(1)-S(0) transition. This is confirmed by comparing the experimental results with numerical calculations based on the density matrix treatment. We also investigate the hydrogen-bonding network surrounding the wild-type (WT)-PYP chromophore in the ground and excited states by comparing its stimulated Raman spectra with those of the E46Q-PYP mutant. We focus on the relative intensity of the Raman band at 1555 cm(-1), which includes both vinyl bond C═C stretching and ring vibrations and is sensitive to the hydrogen-bonding network around the phenolic oxygen of the chromophore. The relative intensity for the WT-PYP decreases after actinic excitation within the 150 fs time resolution and reaches a similar intensity to that for E46Q-PYP. These observations indicate that the WT-PYP hydrogen-bonding network is immediately rearranged in the electronic excited state to form a structure similar to that of E46Q-PYP.

Modulating Native-like Residual Structure in the Fully Denatured State of Photoactive Yellow Protein Affects Its Refolding

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured 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.

Sampling the unfolding pathways towards the signaling state of Photoactive Yellow Protein

When receiving a signal trigger, sensor proteins undergo conformational changes resulting in the formation of a signaling state. Photoactive Yellow Protein (PYP) is a bacterial blue light sensor, 125 amino acids in size, including para-coumaric acid as a chromophore. Upon absorbing a blue-light photon, PYP undergoes a series of rearrangements to form a signaling state. The last step in this process is partial unfolding of the protein, occurring on a sub-millisecond timescale. Molecular simulation can provide detailed insight into the mechanisms underlying protein conformational changes and is complementary to experiments. Studying a protein folding reaction at atomistic resolution with conventional atomistic Molecular Dynamics (MD) is unpractical due to the long time scales involved. These long time scales originate from the presence of local free energy minima from which it is not trivial to escape. Advanced simulations enabled us to investigate the equilibrium characteristics, as well as the dynamical pathways of conformational changes linked to the formation of the signaling state of PYP. Replica exchange MD resulted in the identification of several intermediates during the light induced unfolding. Using these state as input for transition path sampling and subsequent reaction coordinate analysis led to new mechanistic insights in this conformational change. The conformational change starts with the unfolding of a helix in the chromophore binding pocket, followed by the solvent exposure of either the chromophore or glutamate at position 46. Furthermore, our simulations indicate that it is more likely that Glu46 becomes solvent exposed first. To our knowledge this is the first simulation study of unbiased dynamical pathways of a sub-millisecond timescale process of a biologically relevant protein. This work opens up the way for investigating conformational changes in other interesting systems in high detail.

The Transient Accumulation of the Signaling State of Photoactive Yellow Protein Is Controlled by the External pH

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I2′. The pH dependence of the equilibrium between the transient photocycle intermediates I2 and I2′ was investigated. The formation of I2′ from I2 is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I1, I2, and I2′. Their spectra were determined using the extrapolated difference method. I2 and I2′ have electronic absorption spectra, with maxima at 370±5 and 350±5nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I1, I2, and I2′ intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I2′ (∼2ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I2 and I2′ is pH dependent with a pKa of 6.4 and with I2′ the main species above this pKa. Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355nm at a delay of 20ms confirm this pKa value. I2 and I2′ are photoreversed with reversal times of ∼55μs and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pKa of ∼6.1. Photoreversal from I2′ dominates above the pKa. The transient accumulation of I2′, the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k3 for the recovery to the initial dark state also has a pKa of ∼6.3. This equality of the equilibrium and kinetic pKa values is not accidental and suggests that k3 is proportional to [I2′].

Predicting the Signaling State of Photoactive Yellow Protein

As a bacterial blue light sensor the photoactive yellow protein (PYP) undergoes conformational changes upon signal transduction. The absorption of a photon triggers a series of events that are initially localized around the protein chromophore, extends to encompass the whole protein within microseconds, and leads to the formation of the transient pB signaling state. We study the formation of this signaling state pB by molecular simulation and predict its solution structure. Conventional straightforward molecular dynamics is not able to address this formation process due to the long (microsecond) timescales involved, which are (partially) caused by the presence of free energy barriers between the metastable states. To overcome these barriers, we employed the parallel tempering (or replica exchange) method, thus enabling us to predict qualitatively the formation of the PYP signaling state pB. In contrast to the receptor state pG of PYP, the characteristics of this predicted pB structure include a wide open chromophore-binding pocket, with the chromophore and Glu(46) fully solvent-exposed. In addition, loss of alpha-helical structure occurs, caused by the opening motion of the chromophore-binding pocket and the disruptive interaction of the negatively charged Glu(46) with the backbone atoms in the hydrophobic core of the N-terminal cap. Recent NMR experiments agree very well with these predictions.

Short hydrogen bonds in photoactive yellow protein.

Eight high-resolution crystal structures of the ground state of photoactive yellow protein (PYP) solved under a variety of conditions reveal that its chromophore is stabilized by two unusually short hydrogen bonds. Both Tyr42 Oeta and Glu46 Oepsilon are separated from the chromophore phenolate oxygen by less than the sum of their atomic van der Waals radii, 2.6 angstroms. This is characteristic of strong hydrogen bonding, in which hydrogen bonds acquire significant covalent character. The hydrogen bond from the protonated Glu46 to the negatively charged phenolate oxygen is 2.58 +/- 0.01 angstroms in length, while that from Tyr42 is considerably shorter, 2.49 +/- 0.01 angstroms. The E46Q mutant was solved to 0.95 angstroms resolution; the isosteric mutation increased the length of the hydrogen bond from Glx46 to the chromophore by 0.29 +/- 0.01 angstroms to that of an average hydrogen bond, 2.88 +/- 0.01 angstroms. The very short hydrogen bond from Tyr42 explains why mutating this residue has such a severe effect on the ground-state structure and PYP photocycle. The effect of isosteric mutations on the photocycle can be largely explained by the alterations to the length and strength of these hydrogen bonds.

Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.

Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Complete Chemical Structure of Photoactive Yellow Protein: Novel Thioester-Linked 4-Hydroxycinnamyl Chromophore and Photocycle Chemistry

The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.