Photoactive Yellow Protein Research Articles

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.

A Hückel Model for the Excited-State Dynamics of a Protein Chromophore Developed Using Photoelectron Imaging.

ConspectusChemistry can be described as the movement of nuclei within molecules and the concomitant instantaneous change in electronic structure. This idea underpins the central chemical concepts of potential energy surfaces and reaction coordinates. To experimentally capture such chemical change therefore requires methods that can probe both the nuclear and electronic structure simultaneously and on the time scale of atomic motion. In this Account, we show how time-resolved photoelectron imaging can do exactly this and how it can be used to build a detailed and intuitive understanding of the electronic structure and excited-state dynamics of chromophores. The chromophore of the photoactive yellow protein (PYP) is used as a case study. This chromophore contains a para-substituted phenolate anion, where the substituent, R, can be viewed as an acrolein derivative. It is shown that the measured photoelectron angular distribution can be directly related to the electronic structure of the para-substituted phenolate anion. By incrementally considering differing R groups, it is also shown that these photoelectron angular distributions are exquisitely sensitive to the conformational flexibility of R and that when R contains a π-system the excited states of the chromophore can be viewed as a linear combination of the π* molecular orbitals on the phenolate (πPh*) and the R substituent (πR*). Such Hückel treatment shows that the S1 state of the PYP chromophore has predominantly πR* character and that it is essentially the same as the chromophore of the green fluorescent protein (GFP). The S1 excited-state dynamics of the PYP chromophore probed by time-resolved photoelectron imaging clearly reveals both structural (nuclear) dynamics through the energy spectrum and electronic dynamics through the photoelectron angular distributions. Both motions can be accurately assigned using quantum chemical calculations, and these are consistent with the intuitive Hückel treatment presented. The photoactive protein chromophores considered here are examples of where a chemists’ intuitive Hückel view for ground-state chemistry appears to be transferable to the prediction of photochemical excited-state reactivity. While elegant and insightful, such models have limitations, including nonadiabatic dynamics, which is present in a related PYP chromophore, where a fraction of the S1 state population forms a nonvalence (dipole-bound) state of the anion.

Newly identified C-H⋯O hydrogen bond in histidine.

New Cδ-H⋯O histidine hydrogen bonding interactions in various proteins are identified by neutron diffraction and computationally characterized. Neutron diffraction data shows several H-bond motifs with the Cδ-H moiety in histidine side chains, including interactions in β-sheets and with coordinated waters, mostly with histidinium and τ-tautomers. In yellow protein, an active site histidine H-bonds via Cδ-H to a main chain carbonyl while the Cε-H bond coordinates a water molecule. Although the H-bonding ability of Cε-H bonds in histidine have been previously identified, analysis of neutron diffraction structures reveals Cε-H H-bonds in notable active site interactions: for the proximal histidine in myoglobin; a zinc-bound histidine in human carbonic anhydrase II; within the Ser-Asp-His catalytic triad of the trypsin active site; and a histidine in the proton shuttle mechanism of RNase A, in addition to more general roles of coordinating water and forming H-bonds with carbonyl groups in β-sheets within a number of proteins. Properties of these H-bonds were computationally investigated using 5-methylimidazole and 5-methylimidazolium as models for histidine and histidinium. The π- and τ-tautomeric states of 5-methylimidazole were investigated, as both histidine tautomers are observed in the crystal structures. The newly characterized Cε-H⋯O and Cδ-H⋯O model complexes with water and acetone meet the overwhelming majority of IUPAC H-bonding criteria. 5-Methylimidazolium forms complexes that are nearly twice as strong as the respective neutral τ-5-methylimidazole and π-5-methylimidazole complexes. While the τ- and π-tautomers form Cε-H⋯O complexes of similar strength, the τ-Cδ-H⋯O interaction is approximately twice as strong as the π-Cδ-H⋯O interaction. Calculated charges on C-H (and N-H) hydrogens not participating in the H-bond are only slightly perturbed upon complex formation, implying that formation of one H-bond does not diminish the molecule's capacity for further H-bond formation at other sites in the imidazole ring. Overall, findings indicate that the Cδ-H⋯O interaction may be important for β-sheet stability, conformation, interactions with solvent, and mechanisms in the active site. Recognition of C-H bond polarity and hydrogen bonding ability in histidine may improve molecular modeling and provide further insight into the diverse roles of histidine in protein structure-function-dynamics.

Photo-isomerization of the isolated photoactive yellow protein chromophore: what comes before the primary step?

Photoactive proteins typically rely on structural changes in a small chromophore to initiate a biological response. While these changes often involve isomerization as the "primary step", preceding this is an ultrafast relaxation of the molecular framework caused by the sudden change in electronic structure upon photoexcitation. Here, we capture this motion for an isolated model chromophore of the photoactive yellow protein using time-resolved photoelectron imaging. It occurs in <150 fs and is apparent from a spectral shift of ∼70 meV and a change in photoelectron anisotropy. Electronic structure calculations enable the quantitative assignment of the geometric and electronic structure changes to a planar intermediate from which the primary step can then proceed.

Cover Image

AbstractFusions between the Z‐domain and photoactive yellow protein are depicted in yellow. Two of the fusions are not hit by the ray of blue light and remain inactive. The third, in response to blue light irradiation, undergoes conformational changes, allowing for target (Fc) binding (the purple protein). Artwork created by Si Ting Shan of CShan Visuals (https://www.cshan‐visuals.com/). See article by Woloschuk et al., pp. 2359–2372. image

Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Dynamical Behavior of Water; Fluctuation, Reactions and Phase Transitions

Abstract Water dynamical and thermodynamical properties in molecular scale were theoretically investigated in a wide range of temperatures to clarify the physical origin of anomalous water properties. It was found in water that there exist intermittent and collective motions that arise from hydrogen bond network rearrangement. These intermittent motions become more distinctive with temperature decrease and diminish at the glass transition. In a deeply supercooled region, water dynamics shows that a new (“the third”) branch exists in its relaxation and in this branch special defects, H2O1, play a critical role. These defects make water glass transition temperature remarkably low. The intermittent collective motions have significant effects on water phase transitions and chemical reactions. The detailed dynamical mechanisms of the water freezing and the ice melting processes were analyzed. It was found how the embryos of nuclei for these processes are created and grow. The fast proton transfer mechanism in ice was also investigated to find its physical origin. Due to a strong geometrical constraint in ice, the excess proton is not trapped in a deep energy minimum and makes a facile transfer on the small energy barrier surface. As for the auto-dissociation process of water molecules, non-monotonic temperature dependence was theoretically clarified in a wide range of temperatures from ambient to supercritical region. On water roles in biomolecular functions, ion/proton transports and concomitant molecular relaxations were examined in ion-channel, photoactive yellow protein and reaction center.

Few-fs resolution of a photoactive protein traversing a conical intersection.

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago1, conical intersections remain the subject of intense scientific interest2-5. In biology, they have a pivotal role in vision, photosynthesis and DNA stability6. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer7. Spectroscopy can achieve better time resolutions8, but provides indirect structural information. Here we present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved.

The Influence of Electronic Polarization on Nonlinear Optical Spectroscopy.

The environment surrounding a chromophore can dramatically affect the energy absorption and relaxation process, as manifested in optical spectra. Simulations of nonlinear optical spectroscopy, such as two-dimensional electronic spectroscopy (2DES) and transient absorption (TA), will be influenced by the computational model of the environment. We here compare a fixed point charge molecular mechanics model and a quantum mechanical (QM) model of the environment in computed 2DES and TA spectra of Nile red in water and the chromophore of photoactive yellow protein (PYP) in water and protein environments. In addition to simulating these nonlinear optical spectra, we directly juxtapose the computed excitation energy correlation function to the dynamic Stokes shift function often used to analyze environment dynamics. Overall, we find that for the three systems studied here the mutual electronic polarization provided by the QM environment manifests in broader 2DES signals, as well as a larger reorganization energy and a larger static Stokes shift due to stronger coupling between the chromophore and the environment.

All-Optical Switching Demonstrated with Photoactive Yellow Protein Films.

Integrated optics (IO) is a field of photonics which focuses on manufacturing circuits similar to those in integrated electronics, but that work on an optical basis to establish means of faster data transfer and processing. Currently, the biggest task in IO is finding or manufacturing materials with the proper nonlinear optical characteristics to implement as active components in IO circuits. Using biological materials in IO has recently been proposed, the first material to be investigated for this purpose being the protein bacteriorhodopsin; however, since then, other proteins have also been considered, such as the photoactive yellow protein (PYP). In our current work, we directly demonstrate the all-optical switching capabilities of PYP films combined with an IO Mach–Zehnder interferometer (MZI) for the first time. By exploiting photoreactions in the reaction cycle of PYP, we also show how a combination of exciting light beams can introduce an extra degree of freedom to control the operation of the device. Based on our results, we discuss how the special advantages of PYP can be utilized in future IO applications.

Structure-based design of a photoswitchable affibody scaffold.

Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

Effect of Hydrated Ionic Liquid on Photocycle and Dynamics of Photoactive Yellow Protein.

The mechanism by which proteins are solvated in hydrated ionic liquids remains an open question. Herein, the photoexcitation dynamics of photoactive yellow protein dissolved in hydrated choline dihydrogen phosphate (Hy[ch][dhp]) were studied by transient absorption and transient grating spectroscopy. The photocyclic reaction of the protein in Hy[ch][dhp] was similar to that observed in the buffer solution, as confirmed by transient absorption spectroscopy. However, the structural change of the protein during the photocycle in Hy[ch][dhp] was found to be different from that observed in the buffer solution. The known change in the diffusion coefficient of the protein was apparently suppressed in high concentrations of [ch][dhp], plausibly due to stabilization of the secondary structure.

Reversible molecular motional switch based on circular photoactive protein oligomers exhibits unexpected photo-induced contraction

SUMMARYMolecular switches alterable between two stable states by environmental stimuli, such as light and temperature, offer the potential for controlling biological functions. Here, we report a circular photoswitchable protein complex made of multiple protein molecules that can rapidly and reversibly switch with significant conformational changes. The structural and photochromic properties of photoactive yellow protein (PYP) are harnessed to construct circular oligomer PYPs (coPYPs) of desired sizes. Considering the light-induced N-terminal protrusion of monomer PYP, we expected coPYPs would expand upon irradiation, but time-resolved X-ray scattering data reveal that the late intermediate has a pronounced light-induced contraction motion. This work not only provides an approach to engineering a novel protein-based molecular switch based on circular oligomers of well-known protein units but also demonstrates the importance of characterizing the structural dynamics of designed molecular switches.

Skeletal Structure of the Chromophore of Photoactive Yellow Protein in the Excited State Investigated by Ultraviolet Femtosecond Stimulated Raman Spectroscopy.

We studied ultrafast structural dynamics of photoactive yellow protein (PYP) using ultraviolet femtosecond stimulated Raman spectroscopy. By employing the Raman pump and probe pulses in the ultraviolet region, resonantly enhanced, rich vibrational features of the excited-state chromophore were observed in the fingerprint region. In contrast to the marked spectral change reported for the excited-state chromophore in solution, in the protein, all of the observed Raman bands in the fingerprint region did not show any noticeable spectral shifts nor band shape changes during the excited-state lifetime of PYP. This indicates that the significant skeletal change does not occur on the chromophore in the excited state of PYP and that the trans conformation is retained in its lifetime. Based on the femtosecond Raman data of PYP obtained so far, we discuss a comprehensive picture of the excited-state structural dynamics of PYP.

Combined NMR and UV-Vis Spectroscopic Studies of Models for the Hydrogen Bond System in the Active Site of Photoactive Yellow Protein: H-Bond Cooperativity and Medium Effects.

Intramolecular hydrogen bonds in aprotic media were studied by combined (simultaneous) NMR and UV-vis spectroscopy. The species under investigation were anionic and featured single or coupled H-bonds between, for example, carboxylic groups and phenolic oxygen atoms (COO···H···OC)-, among phenolic oxygen atoms (CO···H···OC)-, and hydrogen bond chains between a carboxylic group and two phenolic oxygen atoms (COO···H···(OC)···H···OC)-. The last anion may be regarded as a small molecule model for the hydrogen bond system in the active site of wild-type photoactive yellow protein (PYP) while the others mimic the corresponding H-bonds in site-selective mutants. Proton positions in isolated hydrogen bonds and hydrogen bond chains were assessed by calculations for vacuum conditions and spectroscopically for the two media, CD2Cl2 and the liquefied gas mixture CDClF2/CDF3 at low temperatures. NMR parameters allow for the estimation of time-averaged H-bond geometries, and optical spectra give additional information about geometry distributions. Comparison of the results from the various systems revealed the effects of the formation of hydrogen bond chains and changes of medium conditions on the geometry of individual H-bonds. In particular, the proton in a hydrogen bond to a carboxylic group shifts from the phenolic oxygen atom in the system COO-···H-OC to the carboxylic group in COO-H···(OC)-···H-OC as a result of hydrogen bond formation to the additional phenolic donor. Increase in medium polarity may, however, induce the conversion of a structure of a type COO-H···(OC)-···H-OC to the type COO-···H-(OC)···H-OC. Application of these results obtained from the model systems to PYP suggests that both cooperative effects within the hydrogen bond chain and a low-polarity protein environment are prerequisites for the stabilization of negative charge on the cofactor and hence for the spectral tuning of the photoreceptor.

Open-Source Multi-GPU-Accelerated QM/MM Simulations with AMBER and QUICK.

The quantum mechanics/molecular mechanics (QM/MM) approach is an essential and well-established tool in computational chemistry that has been widely applied in a myriad of biomolecular problems in the literature. In this publication, we report the integration of the QUantum Interaction Computational Kernel (QUICK) program as an engine to perform electronic structure calculations in QM/MM simulations with AMBER. This integration is available through either a file-based interface (FBI) or an application programming interface (API). Since QUICK is an open-source GPU-accelerated code with multi-GPU parallelization, users can take advantage of "free of charge" GPU-acceleration in their QM/MM simulations. In this work, we discuss implementation details and give usage examples. We also investigate energy conservation in typical QM/MM simulations performed at the microcanonical ensemble. Finally, benchmark results for two representative systems in bulk water, the N-methylacetamide (NMA) molecule and the photoactive yellow protein (PYP), show the performance of QM/MM simulations with QUICK and AMBER using a varying number of CPU cores and GPUs. Our results highlight the acceleration obtained from a single or multiple GPUs; we observed speedups of up to 53× between a single GPU vs a single CPU core and of up to 2.6× when comparing four GPUs to a single GPU. Results also reveal speedups of up to 3.5× when the API is used instead of FBI.

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.

Frontiers in Multiscale Modeling of Photoreceptor Proteins.

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments.

A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.

Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Reversible Molecular Motional Switch Based on Circular Photoactive Protein Oligomers: Unexpected Photo-Induced Contraction

Molecular switches alterable between two stable states by environmental stimuli such as light and temperature offer the potential for controlling biological functions. Here, we report a photoswitchable protein complex made of multiple protein molecules that can rapidly and reversibly switch with significant conformational changes. The structural and photochromic properties of photoactive yellow protein (PYP) are harnessed to construct circular oligomer PYPs (coPYPs) of desired sizes. Considering the light-induced N-terminal protrusion of monomer PYP, we expected coPYPs would expand upon irradiation, but time-resolved x-ray scattering data reveal that the late intermediate has a light-induced contraction motion. This work not only provides an approach to engineering a novel protein-based molecular switch based on circular oligomers of a well-known protein unit but also demonstrates the importance of actually characterizing the structural dynamics of designed molecular switches.