Photocycle Of Photoactive Yellow Protein Research Articles

Watching a signaling protein function: What has been learned over four decades of time-resolved studies of photoactive yellow protein.

Photoactive yellow protein (PYP) is a signaling protein whose internal p-coumaric acid chromophore undergoes reversible, light-induced trans-to-cis isomerization, which triggers a sequence of structural changes that ultimately lead to a signaling state. Since its discovery nearly 40 years ago, PYP has attracted much interest and has become one of the most extensively studied proteins found in nature. The method of time-resolved crystallography, pioneered by Keith Moffat, has successfully characterized intermediates in the PYP photocycle at near atomic resolution over 12 decades of time down to the sub-picosecond time scale, allowing one to stitch together a movie and literally watch a protein as it functions. But how close to reality is this movie? To address this question, results from numerous complementary time-resolved techniques including x-ray crystallography, x-ray scattering, and spectroscopy are discussed. Emerging from spectroscopic studies is a general consensus that three time constants are required to model the excited state relaxation, with a highly strained ground-state cis intermediate formed in less than 2.4 ps. Persistent strain drives the sequence of structural transitions that ultimately produce the signaling state. Crystal packing forces produce a restoring force that slows somewhat the rates of interconversion between the intermediates. Moreover, the solvent composition surrounding PYP can influence the number and structures of intermediates as well as the rates at which they interconvert. When chloride is present, the PYP photocycle in a crystal closely tracks that in solution, which suggests the epic movie of the PYP photocycle is indeed based in reality.

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.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4 km long X-ray source, which produces femtosecond, ultra-brilliant and spatially coherent X-ray pulses at megahertz repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological reactions constitute a particularly important class of experiments with biomedical and health related implications. Due to the unusual X-ray pulse structure, such an experiment is challenging. Here we demonstrate how a biological reaction can be followed on ultrafast time scales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. Results connect the previously explored femtosecond PYP dynamics to time scales accessible at synchrotrons. This opens the door to a wide range of time resolved studies at the EuXFEL.

Role of Photoisomerization on the Photodetachment of the Photoactive Yellow Protein Chromophore

The photocycle of photoactive yellow protein (PYP) is initiated by a photoinduced trans-cis isomerization around a C═C bond in the chromophore that lies at the heart of the protein; however, in addition to the desired photochemical pathway, the chromophore can undergo competing electronic relaxation processes. Here we combine gas-phase anion photoelectron spectroscopy and quantum chemistry calculations to investigate how locking the C═C bond in the chromophore controls the competition between these electronic relaxation processes following photoexcitation in the range 400-310 nm. We find evidence to suggest that preventing trans-cis isomerization effectively turns off internal conversion to the ground electronic state and enhances electron emission from the first electronically excited state.

Correction to "Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics".

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to “Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics”Cheolhee Yang, Seong Ok Kim, Yonggwan Kim, So Ri Yun, Jungkweon Choi*, and Hyotcherl Ihee*Cite this: J. Phys. Chem. B 2017, 121, 21, 5461Publication Date (Web):May 17, 2017Publication History Published online17 May 2017Published inissue 1 June 2017https://doi.org/10.1021/acs.jpcb.7b04090Copyright © 2017 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views305Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (134 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

A short history of structure based research on the photocycle of photoactive yellow protein.

The goals of time-resolved macromolecular crystallography are to extract the molecular structures of the reaction intermediates and the reaction dynamics from time-resolved X-ray data alone. To develop the techniques of time-resolved crystallography, biomolecules with special properties are required. The Photoactive Yellow Protein is the most sparkling of these.

Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics.

Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state.

Electronic structure and dynamics of torsion-locked photoactive yellow protein chromophores.

The photocycle of photoactive yellow protein (PYP) begins with small-scale torsional motions of the chromophore leading to large-scale movements of the protein scaffold triggering a biological response. The role of single-bond torsional molecular motions of the chromophore in the initial steps of the PYP photocycle are not fully understood. Here, we employ anion photoelectron spectroscopy measurements and quantum chemistry calculations to investigate the electronic relaxation dynamics following photoexcitation of four model chromophores, para-coumaric acid, its methyl ester, and two analogues with aliphatic bridges hindering torsional motions around the single bonds adjacent to the alkene group. Following direct photoexcitation of S1 at 400 nm, we find that both single bond rotations play a role in steering the PYP chromophore through the S1/S0 conical intersection but that rotation around the single bond between the alkene moiety and the phenoxide group is particularly important. Following photoexcitation of higher lying electronic states in the range 346-310 nm, we find that rotation around the single bond between the alkene and phenoxide groups also plays a key role in the electronic relaxation from higher lying states to the S1 state. These results have potential applications in tuning the photoresponse of photoactive proteins and materials with chromophores based on PYP.

Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila.

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.

Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.

The capacity to respond to environmental changes is crucial to an organism's survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

Watching Proteins Function with Time-Resolved X-ray Diffraction

To understand how proteins function, it is crucial to know the time-ordered sequence of structural changes that occur as they execute their designed function. We recently developed on the BioCARS beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in proteins with 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore [1], and geminate ligand-binding dynamics in hemoglobin [2]. Briefly, a picosecond laser pulse photoexcites a protein and triggers a structural change, which is probed with a suitably delayed picosecond X-ray pulse. This “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue. The mechanistically detailed, near-atomic resolution description of the PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics [3]. This research was supported in part by the Intramural Research Program of the NIH, NIDDK.

Functional Mechanism of the Photoactive Yellow Protein: A Transient Absorption Spectroscopy Perspective

Photoactive yellow protein (PYP) is a photoreceptor from extremophile bacteria Halorhodospira Halophila that swims out of blue light. PYP is supposed to be the responsible for the physiological action. Since the 3D structure of PYP resembles to the PAS domain proteins, PYP is proposed as a model system. The knowledge on how PYP responds to blue light- will shed light on the signal transduction mechanism of the PAS domain proteins. The excitation of blue light triggers the photocycle of PYP through an isomerization of para-coumaric acid chromophore with successive events of the protonation of chromophore, partial-unfolding of protein and resuming of initial state within the seconds of time scale. The partial unfolded state is supposed to be the signaling state. Here, the comprehensive results on the kinetics, dynamics and equilibria of these photocycle intermediates- recently discovered from the transient absorption measurements of the wild type and several key mutants- will be presented. PYP will continue to be the significant part of a newly introduced field of Optogenetics.

Multiscale approach to the determination of the photoactive yellow protein signaling state ensemble.

The nature of the optical cycle of photoactive yellow protein (PYP) makes its elucidation challenging for both experiment and theory. The long transition times render conventional simulation methods ineffective, and yet the short signaling-state lifetime makes experimental data difficult to obtain and interpret. Here, through an innovative combination of computational methods, a prediction and analysis of the biological signaling state of PYP is presented. Coarse-grained modeling and locally scaled diffusion map are first used to obtain a rough bird's-eye view of the free energy landscape of photo-activated PYP. Then all-atom reconstruction, followed by an enhanced sampling scheme; diffusion map-directed-molecular dynamics are used to focus in on the signaling-state region of configuration space and obtain an ensemble of signaling state structures. To the best of our knowledge, this is the first time an all-atom reconstruction from a coarse grained model has been performed in a relatively unexplored region of molecular configuration space. We compare our signaling state prediction with previous computational and more recent experimental results, and the comparison is favorable, which validates the method presented. This approach provides additional insight to understand the PYP photo cycle, and can be applied to other systems for which more direct methods are impractical.

Reply to 'Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein'

Reply to 'Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein'

Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein

Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein

Chromophore Dynamics in the PYP Photocycle from Femtosecond Stimulated Raman Spectroscopy

Femtosecond stimulated Raman spectroscopy (FSRS) is used to examine the structural dynamics of the para-hydroxycinnamic acid (HCA) chromophore during the first 300 ps of the photoactive yellow protein (PYP) photocycle, as the system transitions from its vertically excited state to the early ground state cis intermediate, I0. A downshift in both the C7═C8 and C1═O stretches upon photoexcitation reveals that the chromophore has shifted to an increasingly quinonic form in the excited state, indicating a charge shift from the phenolate moiety toward the C9═O carbonyl, which continues to increase for 170 fs. In addition, there is a downshift in the C9═O carbonyl out-of-plane vibration on an 800 fs time scale as PYP transitions from its excited state to I0, indicating that weakening of the hydrogen bond with Cys69 and out-of-plane rotation of the C9═O carbonyl are key steps leading to photoproduct formation. HOOP intensity increases on a 3 ps time scale during the formation of I0, signifying distortion about the C7═C8 bond. Once on the I0 surface, the C7═C8 and C1═O stretches blue shift, indicating recovery of charge to the phenolate, while persistent intensity in the HOOP and carbonyl out-of-plane modes reveal HCA to be a cissoid structure with significant distortion about the C7═C8 bond and of C9═O out of the molecular plane.

Effect of Hofmeister cosolutes on the photocycle of photoactive yellow protein at moderately alkaline pH

The photocycle of photoactive yellow protein was studied by kinetic absorption spectroscopy from below 100ns to seconds, at moderately alkaline pH, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit of the flash-induced difference spectra provided evidence for five intermediates, including a spectrally silent form before the final recovery of the parent state, but only three with significantly distinct spectra. The calculated intermediate spectra constituted the input for the following spectrotemporal model fit using a sufficiently complex photocycle scheme with reversible transitions. This yielded the rate coefficients of the molecular transitions, the final spectra and the kinetics of the intermediates. Except for the transition between the two red shifted (early) intermediates (pR1 and pR2) and the final photocycle step, all reactions appeared to be reversible. Kosmotropic and chaotropic cosolutes had a systematic effect on the molecular rate coefficients. The largest effect, associated presumably with the exposure of the hydrophobic interior of the protein, accompanies the transition between the second red-shifted and the first blue-shifted intermediate (pR2 and pB1, respectively), i.e. it coincides with the chromophore protonation. The dependence of the rate coefficients on the Hofmeister cosolutes suggests that the conformational change of photoactive yellow protein leading eventually to the most unfolded signaling state takes place in several steps, and starts already with the relaxation after the chromophore isomerization in the microsecond time domain.

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.

Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump–Probe X-ray Solution Scattering

Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.

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.