Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

Determining 3D intermediate structures during the biological action of proteins in real time under ambient conditions is essential for understanding how proteins function. Here we use time-resolved Laue crystallography to extract short-lived intermediate structures and thereby unveil signal transduction in the blue light photoreceptor photoactive yellow protein (PYP) from Halorhodospira halophila. By analyzing a comprehensive set of Laue data during the PYP photocycle (forty-seven time points from one nanosecond to one second), we track all atoms in PYP during its photocycle and directly observe how absorption of a blue light photon by its p-coumaric acid chromophore triggers a reversible photocycle. We identify a complex chemical mechanism characterized by five distinct structural intermediates. Structural changes at the chromophore in the early, red-shifted intermediates are transduced to the exterior of the protein in the late, blue-shifted intermediates through an initial "volume-conserving" isomerization of the chromophore and the progressive disruption of hydrogen bonds between the chromophore and its surrounding binding pocket. These results yield a comprehensive view of the PYP photocycle when seen in the light of previous biophysical studies on the system.     

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.     

Crystallographic structure of a photoreceptor protein at 2.4 A resolution

The first essential step in protein photoreception is the capture and storage of energy from a photon. We have recently identified and isolated, from the purple photoautotrophic bacterium, Ectothiorhodospira halophila, a 13,000-dalton photoactive yellow protein (PYP) that has a photocycle with kinetics similar to sensory rhodopsin and a very high quantum efficiency. To study the structural chemistry of protein photoreception, we determined, refined, and analyzed the crystallographic structure of PYP at 2.4 A resolution and report here that it is composed of two perpendicular antiparallel beta-sheets that enclose the chromophore. Each of the 10 beta-strands of PYP is connected directly to its nearest neighbor with +1 topology. Globally, an asymmetric distribution of side chains places aromatic and acidic side chains in an ellipsoidal band around the chromophore with a cluster of basic side chains on one side. Locally, the electron density maps place an internal lysine and the chromophore in an apparent Schiff base linkage stabilized by a buried glutamate and a tyrosine side chain. To our knowledge, the atomic resolution structure of a protein with a reversible photoisomerization has not been reported previously. Furthermore, PYP may also represent a class of proteins that bind conjugated molecules and interact with a secondary receptor system.     

Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data

We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.     

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: conformational variability and signal transduction

Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per-Arnt-Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 A resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the beta 4-beta 5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the alpha 3-alpha 4 and beta 4-beta 5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and, by comparison to other sensory Per-Arnt-Sim domains, in signal transduction. Because of its direct linkage to a measurable biological output, Ppr-PYP serves as an excellent system for understanding how changes in photocycle dynamics affect signaling by PYPs.     

Folding and signaling share the same pathway in a photoreceptor

The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.     

Primary steps of the photoactive yellow protein: isolated chromophore dynamics and protein directed function

The cycle of the photoactive yellow protein (PYP) has been extensively studied, but the dynamics of the isolated chromophore responsible for transduction is unknown. Here, we present real-time observation of the dynamics of the negatively charged chromophore and detection of intermediates along the path of trans-to-cis isomerization using femtosecond mass selection/electron detachment techniques. The results show that the role of the protein environment is not in the first step of double-bond twisting (barrier crossing) but in directing efficient conversion to the cis-structure and in impeding radical formation within the protein.     

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.     

Restriction of motion of protein side chains during the photocycle of bacteriorhodopsin.

Linear dichroism was measured during the photocycle of bacteriorhodopsin. The anisotropy of the sample was produced by the photoselection method. The measurements on purple membrane fragments embedded in agar gel were performed at room temperature with 200 microseconds time resolution at several wavelengths in the 240- to 550-nm spectral region. The induced anisotropy of the retinal chromophore remained constant after the formation of the photocycle intermediate M. The anisotropy was also time independent at the characteristic peaks of the UV absorption change. These experimental data suggest that the direction of the retinal transition dipole moment remains unchanged. Moreover, the affected aromatic protein side chains also do not show any rotational motion when they are in the perturbed or ground states during the photocycle. Our data render it possible to calculate the restricted range of sudden chromophore rotations that might be coupled to the appearance and decay of the M intermediate.     

Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates.

It is possible, by using Fourier-transform infrared (FTIR) difference spectroscopy, to detect the conformational changes occurring in both the protein and the chromophore of bacteriorhodopsin during the photocycle. In contrast to Raman spectroscopy, a laser is unnecessary and hence the problem of a perturbing probe beam is eliminated. Furthermore, the relatively high signal-to-noise ratio obtainable with FTIR enables measurements to be made in minutes over a large spectral range. In the study reported in this paper, we used this method to examine the state of protonation of the retinylidene Schiff base in light-adapted bR570 and in K, the first intermediate in the photocycle. Resonance Raman spectroscopy provides evidence that bR570 is protonated, but these results have been questioned on the basis of theoretical and experimental grounds. FTIR difference spectral changes in the bR570-to-K transition clearly indicate that bR570 contains a protonated Schiff base. In contrast, the K intermediate displays a Schiff base that is altered but still is associated to some degree with a proton. Because the low-temperature FTIR difference spectrum of bR570 and K is similar to the recently reported low-temperature resonance Raman spectra of bR570 and K [Braiman, M. & Mathies, R. (1982) Proc. Natl. Acad. Sci. USA 79, 403-407], we can assign most, but not all, vibrational changes in the bR570-to-K transition to the chromophore. These results are consistent with a simple model of the first step in the photocycle which involves a movement of the Schiff base proton away from a counterion.     

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis/trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.     

Circular permutation and receptor insertion within green fluorescent proteins

Many areas of biology and biotechnology have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca(2+) in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiological indicators.     

Uncovering the hidden ground state of green fluorescent protein

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.     

Hydrogen bond dynamics in the active site of photoactive yellow protein

Hydrogen bonds play major roles in biological structure and function. Nonetheless, hydrogen-bonded protons are not typically observed by X-ray crystallography, and most structural studies provide limited insight into the conformational plasticity of individual hydrogen bonds or the dynamical coupling present within hydrogen bond networks. We report the NMR detection of the hydrogen-bonded protons donated by Tyr-42 and Glu-46 to the chromophore oxygen in the active site of the bacterial photoreceptor, photoactive yellow protein (PYP). We have used the NMR resonances for these hydrogen bonds to probe their conformational properties and ability to rearrange in response to nearby electronic perturbation. The detection of geometric isotope effects transmitted between the Tyr-42 and Glu-46 hydrogen bonds provides strong evidence for robust coupling of their equilibrium conformations. Incorporation of a modified chromophore containing an electron-withdrawing cyano group to delocalize negative charge from the chromophore oxygen, analogous to the electronic rearrangement detected upon photon absorption, results in a lengthening of the Tyr-42 and Glu-46 hydrogen bonds and an attenuated hydrogen bond coupling. The results herein elucidate fundamental properties of hydrogen bonds within the complex environment of a protein interior. Furthermore, the robust conformational coupling and plasticity of hydrogen bonds observed in the PYP active site may facilitate the larger-scale dynamical coupling and signal transduction inherent to the biological function that PYP has evolved to carry out and may provide a model for other coupled dynamic systems.     

Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts.

Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes in the chromophore and apoprotein that change in intensity or position between light-adapted bacteriorhodopsin and the K and M intermediates in its photocycle and between dark-adapted and light-adapted bacteriorhodopsin. Our infrared measurements provide independent verification of resonance Raman results that in light-adapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is unprotonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most likely interpretation of deuterium shifts of the chromophore hydrogen out-of-plane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of those in light-adapted and dark-adapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.     

A photoactive carotenoid protein acting as light intensity sensor

Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red “active” form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.     

Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site

Phytochromes are photochromic photoreceptors with a bilin chromophore that are found in plants and bacteria. The soil bacterium Agrobacterium tumefaciens contains two genes that code for phytochrome-homologous proteins, termed Agrobacterium phytochrome 1 and 2 (Agp1 and Agp2). To analyze its biochemical and spectral properties, Agp1 was purified from the clone of an E. coli overexpressor. The protein was assembled with the chromophores phycocyanobilin and biliverdin, which is the putative natural chromophore, to photoactive holoprotein species. Like other bacterial phytochromes, Agp1 acts as light-regulated His kinase. The biliverdin adduct of Agp1 represents a previously uncharacterized type of phytochrome photoreceptor, because photoreversion from the far-red absorbing form to the red-absorbing form is very inefficient, a feature that is combined with a rapid dark reversion. Biliverdin bound covalently to the protein; blocking experiments and site-directed mutagenesis identified a Cys at position 20 as the binding site. This particular position is outside the region where plant and some cyanobacterial phytochromes attach their chromophore and thus represents a previously uncharacterized binding site. Sequence comparisons imply that the region around Cys-20 is a ring D binding motif in phytochromes.     

Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13 = C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (approximately 10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within < 3 ps and decays with a time constant of 17 +/- 1 ps to form only BR5.12. No emission in the 650- to 900-nm region can be attributed to the red-absorbing species. Since rotation around C-12 - C-13 and isomerization around C-13 = C-14 are prevented in BR5.12, these results demonstrate that motion in these regions of the retinal is (i) necessary to form the K-like intermediate observed in the native BR-570 photocycle and (ii) not necessary to form a red-absorbing intermediate that has spectral and kinetic properties analogous to those of J-625 in the native BR photocycle. Discussions of the excited and ground electronic state assignments for the intermediate observed in the BR5.12 photoreaction are presented.     

Low-barrier hydrogen bond in photoactive yellow protein

Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is caused by the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified ≈87% (819/942) of the hydrogen positions in PYP and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs and the mechanism of the formation of LBHBs.