pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).     

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.     

Light-induced unfolding of photoactive yellow protein mutant M100L

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.     

Predicting the signaling state of photoactive yellow protein

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

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.     

Photocycle and photoreversal of photoactive yellow protein at alkaline pH: kinetics, intermediates, and equilibria

Since the habitat of Halorhodospira halophila is distinctly alkaline, we investigated the kinetics and intermediates of the photocycle and photoreversal of the photoreceptor photoactive yellow protein (PYP) from pH 8 to 11. SVD analysis of the transient absorption time traces in a broad wavelength range (330-510 nm) shows the presence of three spectrally distinct species (I1, I1', and I2') at pH 10. The spectrum of I1' was obtained in two different ways. The maximal absorption is at 425 nm. I1' probably has a deprotonated chromophore and may be regarded as the alkaline form of I2'. At pH 10, the I1 intermediate decays in approximately 330 micros in part to I1' before I1 and I1' decay further to I2' in approximately 1 ms. From the rise of I2' (approximately 1 ms) to the end of the photocycle, the three intermediates (I1, I1', and I2') remain in equilibrium and decay together to P in approximately 830 ms. Assuming that the spectra of I1, I1', and I2' are pH-independent, their time courses were determined. On the millisecond to second time scale, they are in a pH-dependent equilibrium with a pKa of approximately 9.9. With an increase in pH, the I1 and I1' populations increase at the expense of the amount of I2'. The apparent rate constant for the recovery of P slows with an increase in pH with a pKa of approximately 9.7. The equal pH dependence of this rate and the equilibrium concentrations follows, if we assume that the equilibration rates between the intermediates are much faster than the recovery rate and that the recovery occurs from I2'. The pKa of approximately 9.9 is assigned to the deprotonation of the phenol of the surface-exposed chromophore in the I1'-I2' equilibrium. The I1-I1' equilibrium is pH-independent. Photoreversal experiments at pH 10 with the second flash at 355 nm indicate the presence of only one I2-like intermediate, which we assign on the basis of its lambda(max) value to I2'. After the rapid unresolved photoisomerization to I2'(trans), the reversal pathway back to P involves two sequential steps (60 micros and 3 ms). The amplitude spectra show that I1'(trans) and I1(trans) intermediates participate in this reversal.     

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   

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.     

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.     

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.     

Photoisomerization and photoionization of the photoactive yellow protein chromophore in solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.     

Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction

Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing.     

Proton transfer in the photoreceptors phytochrome and photoactive yellow protein.

Light-induced activation of the photoreceptors phytochrome and photoactive yellow protein (PYP) is accompanied by protonation changes of the respective chromophores and key residues in the protein moiety. For both systems, proton exchange with the external medium could be observed with pH electrode measurements and with UV-visible absorption spectroscopy using appropriate pH indicator dyes. From these signals, the stoichiometry of proton release and uptake, respectively, was determined by different calibration procedures which will be presented and discussed. Kinetic information on these processes is only available from time-resolved measurements with pH indicator dyes. Vibrational spectroscopy methods such as Fourier transform infrared spectroscopy and resonance Raman scattering provided information on the protonation state of individual functional groups suggesting that internal proton transfer processes are involved as well. Deuterium kinetic isotope effects that occurred in the Pr --> Pfr phototransformation of the bacteriophytochromes Cph1 and Agp1 were consistent with proton transfer reactions as rate-limiting steps. In contrast, the apparent rate constants in the photocycle of PYP exhibited only small kinetic isotope effects that could not be interpreted conclusively. Possible mechanisms of proton transfer in the activation of phytochrome and PYP will be discussed.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Absorption spectra of photoactive yellow protein chromophores in vacuum

The absorption spectra of two photoactive yellow protein model chromophores have been measured in vacuum using an electrostatic ion storage ring. The absorption spectrum of the isolated chromophore is an important reference for deducing the influence of the protein environment on the electronic energy levels of the chromophore and separating the intrinsic properties of the chromophore from properties induced by the protein environment. In vacuum the deprotonated trans-thiophenyl-p-coumarate model chromophore has an absorption maximum at 460 nm, whereas the photoactive yellow protein absorbs maximally at 446 nm. The protein environment thus only slightly blue-shifts the absorption. In contrast, the absorption of the model chromophore in aqueous solution is significantly blue-shifted (lambda(max) = 395 nm). A deprotonated trans-p-coumaric acid has also been studied to elucidate the effect of thioester formation and phenol deprotonation. The sum of these two changes on the chromophore induces a red shift both in vacuum and in aqueous solution.     

Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering

It is widely accepted that PYP undergoes global structural changes during the formation of the biologically active intermediate PYP(M). High-angle solution x-ray scattering experiments were performed using PYP variants that lacked the N-terminal 6-, 15-, or 23-amino-acid residues (T6, T15, and T23, respectively) to clarify these structural changes. The scattering profile of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 A(-1) < Q < 0.8 A(-1)). The intensities and positions of the peaks were systematically changed as a result of the N-terminal truncations. These observations and the agreement between the observed scattering profiles and the calculated profiles based on the crystal structure confirm that the high-angle scattering profiles were caused by intramolecular interference and that the structure of the chromophore-binding domain was not affected by the N-terminal truncations. The profiles of the PYP(M) intermediates of the N-terminally truncated PYP variants were significantly different from the profiles of the dark states of these proteins, indicating that substantial conformational rearrangements occur within the chromophore-binding domain during the formation of PYP(M). By use of molecular fluctuation analysis, structural models of the chromophore-binding region of PYP(M) were constructed to reproduce the observed profile of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting beta4 and beta5, and the deformation of the alpha4 helix. High-angle x-ray scattering with molecular fluctuation simulation allows us to derive the structural properties of the transient state of a protein in solution.     

Effect of organic anions on the photoreaction of photoactive yellow protein

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.     

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.     

Light-induced global conformational change of photoactive yellow protein in solution

The light-induced global conformational change of photoactive yellow protein was directly observed by small-angle X-ray scattering (SAXS). The N-terminal 6, 15, or 23 amino acid residues were enzymatically truncated (T6, T15, or T23, respectively), and their near-UV intermediates were accumulated under continuous illumination for SAXS measurements. The Kratky plot demonstrated that illumination induced partial loss of globularity. The change in globularity was marked in T6 but very small in T15 and T23, suggesting that structural change in positions 7-15 mainly reduces the globularity. The radius of gyration (R(g)) estimated by Guinier plot was increased by 1.1 A for T6 and 0.7 A for T15 and T23 upon illumination. As T23 lacks most of the N-terminal loop, structural change in the main part composed of the PAS core, helical connector, and beta-scaffold caused an increase of R(g) by 0.7 A. The structural change of positions 7-15 caused an additional increase by 0.4 A. The decrease of R(g) upon truncation of positions 7-15 for dark state was 0.3 A, while that for the intermediate was 0.7 A, suggesting that this region moves outward on formation of the intermediate. These results indicate that a light-induced structural change of PYP takes place in the main part and N-terminal 15 amino acid residues. The former induces only dimensional increase, but the latter results in additional change in shape.