Mimic of photocycle by a protein folding reaction in photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.     

Initial events in the photocycle of photoactive yellow protein

The light-induced isomerization of a double bond is the key event that allows the conversion of light energy into a structural change in photoactive proteins for many light-mediated biological processes, such as vision, photosynthesis, photomorphogenesis, and photo movement. Cofactors such as retinals, linear tetrapyrroles, and 4-hydroxy-cinnamic acid have been selected by nature that provide the essential double bond to transduce the light signal into a conformational change and eventually, a physiological response. Here we report the first events after light excitation of the latter chromophore, containing a single ethylene double bond, in a low temperature crystallographic study of the photoactive yellow protein. We measured experimental phases to overcome possible model bias, corrected for minimized radiation damage, and measured absorption spectra of crystals to analyze the photoproducts formed. The data show a mechanism for the light activation of photoactive yellow protein, where the energy to drive the remainder of the conformational changes is stored in a slightly strained but fully cis-chromophore configuration. In addition, our data indicate a role for backbone rearrangements during the very early structural events.     

Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.

The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.     

Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila.

We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.     

Protonation states and pH titration in the photocycle of photoactive yellow protein

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.     

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.     

Light induces destabilization of photoactive yellow protein

To understand the effect of visible light on the stability of photoactive yellow protein (PYP), urea denaturation experiments were performed with PYP in the dark and with PYP(M) under continuous illumination. The urea concentrations at the midpoint of denaturation were 5.26 +/- 0.29 and 3.77 +/- 0.19 M for PYP and PYP(M), respectively, in 100 mM acetate buffer, and 5.26 +/- 0.24 and 4.11 +/- 0.12 M for PYP and PYP(M), respectively, in 100 mM citrate buffer. The free energy change upon denaturation (DeltaG(D)(H2O)), obtained from the denaturation curve, was 11.0 +/- 0.4 and 7.6 +/- 0.2 kcal/mol for PYP and PYP(M), respectively, in acetate buffer, and 11.5 +/- 0.3 and 7.8 +/- 0.1 kcal/mol for PYP and PYP(M), respectively, in citrate buffer. Even though the DeltaG(D)(H2O) value for PYP(M) is almost identical in the two buffer systems, the urea concentration at the midpoint of denaturation is lower in acetate buffer than in citrate buffer. Although their CD spectra indicate that the protein conformations of the denatured states of PYP and PYP(M) are indistinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interconvertible through PYP and PYP(M). Therefore, the free energy difference between PYP and PYP(M) is 3.4-3.7 kcal/mol for the protein moiety, plus the additional contribution from the difference in configuration of the chromophore.     

Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy

Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I(0) and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.     

Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein.

Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.     

Amino acids in the N-terminal region regulate the photocycle of photoactive yellow protein.

The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied. Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE. They were readily separated by DEAE-Sepharose column chromatography. Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23). The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively. The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively. These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.     

Structural role of tyrosine 98 in photoactive yellow protein: effects on fluorescence, gateway, and photocycle recovery

We have recently shown that the Y98Q mutant of PYP has a major effect on the photocycle kinetics ( approximately 40 times slower recovery). We have now determined the crystal structure of Y98Q at 2.2 A resolution to reveal the role of residue Y98 in the PYP photocycle. Although the overall structure is very similar to that of WT, we observed two major effects of the mutation. One obvious consequence is a conformational change of the beta4-beta5 loop, which includes a repositioning of residue M100. It had previously been shown that the photocycle is slowed by as much as 3 orders of magnitude when residue M100 is substituted or when the conformation is altered as in Rhodocista centenaria PYP. To investigate whether the altered photocycle of Y98Q is due to this repositioning of M100 or is caused by an effect unrelated to M100, we determined the dark recovery kinetics of the Y98Q/M100A mutant. We find the recovery kinetics to be very similar to the M100A single mutant kinetics and therefore conclude that the slower recovery kinetics in Y98Q are most likely due to repositioning of M100. In addition, we find that other substitutions at position 98 (Y98W, Y98L, and Y98A) have differing effects on the photocycle recovery, presumably due to a variable distortion of the beta4-beta5 loop. The second effect of the Y98Q mutation is a repositioning of R52, which is thought to interact with Y98 in WT PYP and now forms new interactions with residues Q99 and Q56. To determine the role of R52, we also characterized an R52A/M100A double mutant and found that the effects on the recovery kinetics ( approximately 2000 slower recovery than WT) are due to unrelated events in the photocycle. Since the Y98Q/M100A recovery kinetics are more similar to those of M100 than R52A/M100A, we conclude that the repositioning of R52, caused by the Y98Q mutation, does not affect the dark state recovery. In addition, it has been proposed that Y98 and P68 are "gateway residues" between which the chromophore must pass during isomerization. We tested the recovery kinetics of mutant P68A and found that, although the gateway may be important for photocycle initiation, its role in recovery to the ground state is minimal.     

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.     

Tryptophan fluorescence monitors structural changes accompanying signalling state formation in the photocycle of photoactive yellow protein.

Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira)[space]halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central [small beta]-sheet. This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap. The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues. Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum. Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases. These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore. The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119. At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced. On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG). This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.     

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.     

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

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

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.     

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

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

Proline 54 trans-cis isomerization is responsible for the kinetic partitioning at the last-step photocycle of photoactive yellow protein

Photoactive yellow protein (PYP), a blue-light photoreceptor for Ectothiorhodospira halophila, has provided a unique system for studying protein folding that is coupled with a photocycle. Upon receptor activation by blue light, PYP proceeds through a photocycle that includes a partially folded signaling state. The last-step photocycle is a thermal recovery reaction from the signaling state to the native state. Bi-exponential kinetics had been observed for the last-step photocycle; however, the slow phase of the bi-exponential kinetics has not been extensively studied. Here we analyzed both fast and slow phases of the last-step photocycle in PYP. From the analysis of the denaturant dependence of the fast and slow phases, we found that the last-step photocycle proceeds through parallel channels of the folding pathway. The burial of the solvent-accessible area was responsible for the transition state of the fast phase, while structural rearrangement from the compact state to the native state was responsible for the transition state of the slow phase. The photocycle of PYP was linked to the thermodynamic cycle that includes both unfolding and refolding of the fast- and slow-phase intermediates. In order to test the hypothesis of proline-limited folding for the slow phase, we constructed two proline mutants: P54A and P68A. We found that only a single phase of the last-step photocycle was observed in P54A. This suggests that there is a low energy barrier between trans to cis conformation in P54 in the light-induced state of PYP, and the resulting cis conformation of P54 generates a slow-phase kinetic trap during the photocycle-coupled folding pathway of PYP.     

Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants.

Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues.     

Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.