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.

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.

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.

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy.

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.

Roles of amino acid residues near the chromophore of photoactive yellow protein.

To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.

Vortex glass transition and quantum vortex liquid at low temperature in a thick a- MoxSi1-x film.

We present measurements of ac complex resistivity, as well as dc resistivity, for a thick amorphous MoxSi1-x film at low temperatures ( T>0.04 K) in various constant fields B. We find that the vortex glass transition (VGT) persists down to T approximately 0.04Tc0 up to B approximately 0.9Bc2(0), where Tc0 and Bc2(0) are the mean-field transition temperature and upper critical field at T = 0, respectively. In the limit T-->0, the VGT line Bg(T) extrapolates to a field below Bc2(0), while the dc resistivity rho(T) tends to the finite nonzero value in fields just above Bg(0). These results indicate the presence of a metallic quantum vortex liquid at T = 0 in the regime Bg(0)

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.

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.

Effect of anion binding on iodopsin studied by low-temperature fourier transform infrared spectroscopy.

The effect of anion binding on iodopsin, the chicken red-sensitive cone visual pigment, was studied by measurements of the Fourier transform infrared spectra of chloride- and nitrate-bound forms of iodopsin at 77 K. In addition to the blue shift of the absorption maximum upon substituting nitrate for chloride, the C=C stretching vibrations of iodopsin and its photoproducts were upshifted 5-6 cm(-)(1). The C=NH and C=ND stretching vibrations were the same in wavenumber between the chloride- and nitrate-bound forms, indicating that the binding of either chloride or nitrate has no effect on the interaction between the protonated Schiff base and the counterion. The vibrational bands of iodopsin in the fingerprint and the hydrogen out-of-plane wagging regions were insensitive to anion substitution, suggesting that local chromophore interactions with the anions are not crucial for the absorption spectral shift. In contrast, bathoiodopsin in the chloride-bound form exhibited an intense C(14)H wagging mode, whose intensity was considerably weakened upon substitution of nitrate for chloride. These results suggest that binding of chloride changes the environment near the C(14) position of the chromophore, which could be one of the factors in the thermal reverse reaction of bathoiodopsin to iodopsin in the chloride-bound form.

Presence of two rhodopsin intermediates responsible for transducin activation.

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.

Photochemical and biochemical properties of chicken blue-sensitive cone visual pigment.

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.

The last phase of the reprotonation switch in bacteriorhodopsin: the transition between the M-type and the N-type protein conformation depends on hydration.

In order to elucidate the mechanism of the reprotonation switch of bacteriorhodopsin, the protein conformation of the M intermediate of the D96N mutant was examined at various hydration conditions by X-ray diffraction and FTIR spectroscopy. We observed two distinct protein conformations at different levels of hydration. One is like in the N photointermediate, although in this case with an unprotonated Schiff base. It is stabilized in highly hydrated samples. The other is a protein conformation identical to that in the normal M intermediate of wild-type bacteriorhodopsin, which is stabilized in partially dehydrated samples. The hydration dependence of the structural transition between the M-type and the N-type conformations suggests that there is a change in the binding of water at the cytoplasmic surface. Thus, more water molecules bind in the N-type structure than in the M-type. This is consistent with the idea that the conformational change from the M-type to the N-type corresponds to the opening of the proton channel to the cytoplasmic surface by tilt of the cytoplasmic end of helix F, and that this is required for proton transfer from Asp-96 to the retinal Schiff base.

Functional expression and site-directed mutagenesis of photoactive yellow protein.

The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

Evidence for proton transfer from Glu-46 to the chromophore during the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 degrees C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 --> Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Chromophore configuration of iodopsin and its photoproducts formed at low temperatures.

The photochemical reactions of iodopsin at low temperatures were investigated by a combination of absorption spectroscopy and chromophore extraction to show the formation of isomeric photoproducts other than the all-trans intermediates. We first confirmed that the chromophore in iodopsin is an 11-cis-retinal. Next, iodopsin samples were irradiated with light of different wavelengths at selected temperatures ranging from -190 to 0 degrees C, and their retinylidene chromophores were extracted as oximes after warming the sample to 0 degree C. The isomeric composition of the extracted chromophores was analyzed by high-performance liquid chromatography. It was confirmed that bathoiodopsin produced at -190 degrees C has an all-trans chromophore, but a considerable amount of its chromophore thermally reisomerizes to the 11-cis form upon warming. Photoproducts formerly assigned as lumi- and metaiodopsins [Yoshizawa, T., & Wald, G. (1967) Nature 214, 566-571; Hubbard, R., & Kropf, A. (1959) Nature 183, 448-450] were produced by extensive irradiation of iodopsin at -80 and -40 degrees C, respectively, with red light, but their chromophores were identified to be 7-cis-retinals instead of all-trans-retinals. Thus these photoproducts are artificial byproducts formed as a result of photon absorption by all-trans intermediates. The absorption spectrum of the 7-cis product formed from bovine rhodopsin shows no spectral shift when it is warmed from -80 to 0 degrees C, but the spectrum of 7-cis species formed from iodopsin shifted about 40 nm to the blue at a transition temperature of -60 degrees C. This result indicates a unique chromophore-opsin interaction in iodopsin. Four all-trans intermediates of iodopsin were identified above -80 degrees C under irradiation conditions in which no 7-cis products accumulated. Their absorption maxima were estimated to be approximately 570, approximately 530, approximately 470, and approximately 380 nm. These species should correspond to BL-iodopsin, lumiiodopsin, metaiodopsin I, and metaiodopsin II, respectively, assigned by room temperature laser photolysis [Shichida, Y., Okada, T., Kandori, H., Fukada, Y., & Yoshizawa, T. (1993) Biochemistry 32, 10832-10838].

Photoreaction cycle of photoactive yellow protein from Ectothiorhodospira halophila studied by low-temperature spectroscopy.

The photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila was studied by low-temperature spectroscopy. Irradiation of PYP at -190 degrees C produced a photo-steady-state mixture composed of bathochromic and hypsochromic photoproducts (PYPB and PYPH). Upon warming, PYPH was thermally converted to a slightly blue-shifted intermediate (PYPHL) above -150 degrees C and then to a red-shifted one (PYPL) above -80 degrees C. PYPB was thermally converted to the blue-shifted intermediate (PYPBL) above -180 degrees C and then to PYPL above -90 degrees C. PYPL thermally reverted to PYP above -50 degrees C, completing the photocycle. The spectral properties of PYPL formed at low temperature suggest that it corresponds to the red-shifted photoproduct detected in the nano- to microsecond time scale at room temperature (A465). The absolute absorption spectra of PYPH, PYPB, and PYPL were estimated, and their absorption maxima were determined to be 442 and 489 nm at -190 degrees C and 456 nm at -80 degrees C, respectively. Although a near-UV intermediate (A355) is observed in the recovery process of PYP from A465 at room temperature, it was not detected at low temperatures, probably due to the effects of temperature and the presence of glycerol. A scheme of the photocycle of PYP is presented.

Structure around C6-C7 bond of the chromophore in bathorhodopsin: low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs.

To elucidate the structural changes near the beta-ionone ring region of the chromophore during the photobleaching process of rhodopsin, the photochemical and subsequent thermal reactions of rhodopsin analogs, whose retinylidene chromophores were fixed in a 6s-cis form with a five-membered ring (6,5-rhodopsin) and a seven-membered ring (6,7-rhodopsin), respectively, were investigated by low-temperature spectroscopy. Like rhodopsin, both the rhodopsin analogs convert to the respective batho-intermediates upon absorption of light at -190 degrees C. The extinction coefficient of batho-intermediate of 6,5-rhodopsin is similar to that of bathorhodopsin, while that of 6,7-rhodopsin is considerably smaller than that of bathorhodopsin. Like bathorhodopsin, the batho-intermediate of 6,5-rhodopsin directly converts to lumi-intermediate, while that of 6,7-rhodopsin first converts to a blue-shifted intermediate and then to lumi-intermediate. These results strongly suggest that the structure around the beta-ionone ring region of the bathorhodopsin chromophore resembles 6,5-retinal rather than 6,7-retinal. From the comparison of the structural features among retinal, 6,5-retinal, and 6,7-retinal, a possible conformation around C6-C7 bond of the bathorhodopsin chromophore is discussed.

Effect of chloride on the thermal reverse reaction of intermediates of iodopsin.

Among the intermediates in the bleaching process of iodopsin, a chicken red-sensitive cone visual pigment, the batho and meta I intermediates (batho and meta I) formed at low temperatures revert to the original iodopsin by thermal reactions [Yoshizawa & Wald (1967) Nature 214, 566-571; Imamoto, Imai, Yoshizawa, & Shichida (1994) FEBS Lett. 354, 165-168]. In order to elucidate the relationship between Cl- binding to iodopsin and these reverse reactions, we have prepared a sample of iodopsin whose Cl(-)-binding site is vacant (anion-unbound iodopsin) and compared the thermal reactions of its batho and meta I intermediates with those of Cl(-)-bound (native) and nitrate-bound iodopsins. The reverse reaction from batho is observed in both Cl(-)-bound and anion-unbound iodopsins, while the reaction from meta I is observed only in Cl(-)-bound iodopsin. These results indicate that Cl- binding is indispensable for the reverse reaction from meta I, but not from batho. The reverse reaction from meta I has been further investigated as a function of Cl- concentration, and the dissociation constant of Cl- in meta I is estimated to be approximately 20 mM. This value is about 200 times larger than that of iodopsin (0.1 mM), and close to the physiological Cl- concentration in photoreceptor cells, suggesting that Cl- could be released from the protein moiety during the bleaching of iodopsin.

Reconstitution photoactive yellow protein from apoprotein and p-coumaric acid derivatives.

We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p-coumaric acid derivatives. The addition of p-coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p-coumaryl thiophenyl ester or p-coumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p-coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.