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Originally published In Press as doi:10.1074/jbc.M506403200 on December 20, 2005

J. Biol. Chem., Vol. 281, Issue 7, 4318-4325, February 17, 2006
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pH-dependent Equilibrium between Long Lived Near-UV Intermediates of Photoactive Yellow Protein*

Nobutaka Shimizu1, Yasushi Imamoto2, Miki Harigai, Hironari Kamikubo, Yoichi Yamazaki, and Mikio Kataoka

From the Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

Received for publication, June 13, 2005 , and in revised form, December 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The long lived intermediate (signaling state) of photoactive yellow protein (PYPM), which is formed in the photocycle, was characterized at various pHs. PYPM at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYPMacid) and alkaline form (PYPMalkali) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pKa was 6.4. Content of {alpha}- and/or beta-structure of PYPMacid was significantly greater than PYPMalkali 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 PYPMacid was smaller than that of PYPMalkali. The vibrational mode at 1747 cm-1 of protonated Glu-46 was found as a small band for PYPMacid but not for PYPMalkali, suggesting that Glu-46 remains partially protonated in PYPMacid, whereas it is fully deprotonated in PYPMalkali. Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYPMacid was 15.7 Å, whereas for PYPMalkali it was 16.2 Å. These results indicate that PYPMacid assumes a more ordered and compact structure than PYPMalkali. Binding of citrate shifts this equilibrium toward PYPMalkali. UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYPMacid, indicating that the mutation shifts this equilibrium toward PYPMacid. Alterations in the nature of PYPM by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYPMacid and PYPMalkali.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Photoactive yellow protein (PYP)3 (1) is a water-soluble photoreceptor protein suggested to be involved in the negative phototaxis of Halorhodospira halophila (2). PYP is an attractive model protein for a common signaling mechanism because it is a structural prototype of the PAS domain superfamily, most of which are involved in the biological sensing (3). PYP is composed of 125 amino acid residues and the p-coumaric acid chromophore linking to the cysteine residue by a thioester bond (4-6). In the dark state, the phenolic oxygen of the chromophore is deprotonated (5) and hydrogen bonded with protonated Tyr-42 and Glu-46 (see Fig. 1), resulting in the yellow color. Photon absorption by the chromophore initiates the photocycle of PYP (7-11). The primary photochemical event of PYP is the trans-cis isomerization of the chromophore (12, 13), like bacterial retinal proteins. Several intermediates are then formed, followed by a return to the dark state in 100 ms under physiological conditions. During the photocycle, photon energy stored in the twisted chromophore is released to the protein moiety to form the putative signaling state (called PYPM, I2, or pB). Because of a largely blue-shifted absorption spectrum with respect to the dark state, as well as the long lifetime (~100 ms), PYPM is easily detectable by UV-visible spectroscopy. This spectral blue shift is considered to be caused by protonation of the phenolic oxygen of the chromophore (5, 14).

Infrared spectroscopy demonstrated that PYPM is formed from its precursor (PYPL,I1, or pR) in two steps (15, 16). First, a proton at Glu-46 is transferred to the chromophore, but the global conformational change of the protein moiety does not take place at this stage, as shown by a small absorbance change of the amide mode. This intermediate (pB') has a protonated chromophore, but the chromophore binding site is considered to be preserved. Second, a global conformational change takes place to form PYPM. As the lifetime of pB' (~1 ms) is not markedly different from pR (~100 µs) and the absorption spectra of PYPM and pB' are similar, isolation of pB' has been difficult in steady-state measurement. Recently, the presence of pB' between PYPL and PYPM has been established by time-resolved UV-visible spectroscopy and Raman spectroscopy (17-19).

Although the signaling pathway of the PYP system has not yet been identified, detailed characterization of long lived intermediates (signaling state) is essential for understanding the signal transduction mechanism of PYP. We have demonstrated extensive conformational change upon formation of PYPM. The dimension of PYPM is significantly larger than the dark state as shown by ~1 Å increase of Rg (20, 21) and PYPM is in a partially unfolded state (22). The N-terminal cap and the central part undergo a substantial structural change (21, 23); however, the cause of this large conformational change has not yet been determined.

Accumulated evidence suggests that properties of PYPM are highly pH-sensitive. The extent of conformational change evaluated by the difference FTIR spectra under acidic conditions (24, 25) is significantly smaller than at neutral pH (15, 23). The decay rate constant of PYPM is reduced by acidification, and the relationship between rate constant and pH agrees with the Henderson-Hasselbalch equation with a pKa of 6.4 (26). Proton uptake per PYPM formation is also represented by the Henderson-Hasselbalch equation with a pKa of 6.6 (27).

On the other hand, we have reported that PYPM substantially alters its nature, depending on the buffer system (20). Absorption spectra as well as the lifetime of PYPM at acidic pH are affected by multivalent organic ions, such as citrate. Mutation of amino acids nearby the chromophore also affects properties of PYPM. The absorption maximum of E46QM is 11-nm red-shifted (28). The protein conformational change for formation of E46QM is significantly smaller than wild type (16, 29), suggesting that the photocycle of E46Q may be different from that of wild type. To study whether there is a simple and consistent explanation for the variety of signaling states or whether each phenomenon has an individual specific reason, the spectroscopic properties of PYPM were studied in detail under various conditions using UV-visible spectroscopy, CD spectroscopy, FTIR spectroscopy, and small angle x-ray scattering measurements. The results are explained by the equilibrium between two states of PYPM.


Figure 1
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  		FIGURE 1.
Chromophore (left) and overall fold (right) of PYP (PDB code, 2phy [PDB] ). This model was drawn by PyMOL (Delano Scientific, San Carlos, CA).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Preparation—Wild type PYP and E46Q were prepared as described previously (30). For UV-visible and CD spectroscopy, PYP or mutant was suspended in 10 mM TAPS buffer (pH 8.5-8.0), 10 mM MOPS buffer (pH 8.0-6.5), or 10 mM MES buffer (pH 6.5-4.0) containing 200 mM NaCl (Dojindo, Kumamoto, Japan). To investigate the effect of binding of citrate to PYPM, 50 mM (see Fig. 6) or 1 M (see Fig. 7) sodium citrate was added instead of NaCl (sample concentration ~0.15 mg/ml). For SAXS measurements, samples at pH 5.0 and 8.0 were concentrated to 2-10 mg/ml with an ultrafiltration membrane (Centricon YM10, Millipore, Billerica, MA). For FTIR measurement, PYP and E46Q were desalted, lyophilized, and resuspended in 100 mM MES buffer (pH 5.0) or TAPS buffer (pH 8.0) containing 200 mM NaCl at a concentration of 150 mg/ml.

UV-visible Spectroscopy—Absorption spectra in the dark and in the photosteady state were obtained by a multichannel CCD/fiber optic spectroscopy system (S2000 system, Ocean Optics, Dunedin, FL) (23). For steady-state measurements, the sample was irradiated with a yellow light obtained from a 150-W cold light source (HL150, HOYA-Schott, Tokyo, Japan) and a Y43 cutoff filter (>410 nm, Asahi Techno Glass, Chiba, Japan). For time-resolved measurements, the sample was excited with a yellow flash obtained with a short arc xenon flash lamp (SA200, Nissin Electronic, Tokyo, Japan) and a Y43 filter. Absorption spectra of PYPM were calculated by subtracting dark spectra from photosteady state spectra (31). At pH > 6, contribution of the small amount of PYPL was also subtracted, as reported previously (31).

CD Spectroscopy—Far-UV CD spectra of dark state and photosteady state were measured using a J-725 circular dichroism spectropolarimeter (JASCO, Tokyo, Japan). Temperature was maintained at 5 °C with a PYC-347WI Peltier device (JASCO). The sample cell, with a 1-mm light path length, was irradiated at an angle of 30° using a HOYA-Schott HL150 light source and an Y43 cutoff filter. To protect the detector, the band pass filter (214FS10-25, 210 nm < {lambda} < 233 nm, Tmax = 14%, Andover Corporation, Salem, NH) was set in front of the detector window. It was confirmed that the artifact on CD signal caused by band-bass filter was negligible in this setup (23).

The photosteady state mixture was composed of PYP, PYPL, and PYPM. The CD spectrum of PYPM was calculated assuming that the CD spectrum of PYPL is indistinguishable from that of PYP because FTIR measurements demonstrate that global structural change takes place at PYPM (32). The fraction of PYPM in the photosteady state mixture was estimated by absorption spectroscopy using an S2000 system under the same irradiation conditions as CD spectroscopy.

FTIR Spectroscopy—Difference FTIR spectra between photosteady state and dark state were measured using an FTS6000 Fourier transform infrared spectrometer (23). The sample was put into a CaF2 cell with a 7.2 µm light path length (23). Irradiation light at 436 nm (FWHM = 10 nm) was obtained by an optical interference filter (43161, Edmund Scientific, Barrington, NJ) and a Hoya-Schott HL150 light source.

SAXS Measurements—Small angle x-ray scattering measurements were carried out at BL-10C (Photon Factory, Tsukuba, Japan (20, 21)). The exposure time was 5 min. Three to five sets of independent measurements were averaged to improve the signal to noise ratio. Temperature of the cell was maintained at 5 °C. Rg and I(0) were obtained by Guinier approximation in the small angle region (33, 34),

Formula(Eq. 1)
where Q = 4{pi}sin{theta}/{lambda} is the amplitude of the scattering vector, I(Q) is the scattering intensity at Q,2{theta} is the scattering angle, {lambda} is the wavelength of the x-ray (1.488 Å), I(0) is the scattering intensity at Q = 0, and Rg is the radius of gyration. The sample was irradiated with yellow light obtained using a 1000-W tungsten-halogen lamp (HILUX-HR, Tokyo Master, Japan) and Y43 cutoff filter. The scattering profiles of photoproducts were calculated using scattering patterns of the photosteady state and dark state based on the fraction estimated by absorption spectroscopy.


Figure 2
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  		FIGURE 2.
UV-visible absorption spectra for PYP and PYPM at various pH. a, absorption spectra dark state PYP at pH 8.5-4.0 with an interval of 0.5. Spectra were measured at 5 °C. b, absorption spectra of PYPM at pH 8.5-4.0. pH values are indicated in the figure. c, absorbance at 395 nm for PYPM was plotted against pH and fitted using the Henderson-Hasselbalch equation.

 
pH Titration—Absorbance at 395 nm (see Fig. 2c) or the ellipticity at 222 nm (see Fig. 3b) of PYPM was plotted against pH. Data were fitted according to Henderson-Hasselbalch equation,

Formula 2(Eq. 2)
where k0 and k1 are a base value and amplitude of the titration curve, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
pH-dependent Spectral Change of PYPM—Fig. 2a shows the absorption spectra of PYP suspended in the buffer at pH 4.0-8.5 in the dark. The absorption spectrum of PYP is independent of pH in this pH region, although it is bleached at pH < 4.1 (pKa = 3) (35, 36). Then samples at various pH values were irradiated by continuous yellow light (>410 nm), and the absorption spectra of the photosteady state were recorded. As the photosteady state is in equilibrium between PYP, PYPL, and PYPM (31, 37), the absorption spectrum of PYPM at each pH was calculated by subtracting the absorption spectra of dark state and PYPL from that of the photosteady state, so that the contributions of PYP and PYPL were cancelled (31). Calculated absorption spectra of PYPM at pH 8.5-4.0 are shown in Fig. 2b. The PYPM spectrum at pH 8.5 had a broad shape with the shoulder at around 330 nm ({lambda}max = 356 nm). Upon acidification, the spectrum was red-shifted ({lambda}max = 367 nm), and the shoulder disappeared. The pH-dependent spectral change of PYPM at pH 8.5-4.0 is virtually a two-state transition with a clear isosbestic point at 362 nm. The absorbance change at 395 nm was plotted against pH (Fig. 2c). Absorbance at 395 nm was increased from pH 8.5 to 5.0, but decreased at pH < 5, suggesting that a part of PYPM is denatured at low pH. The absorbance change was fitted by the Henderson-Hasselbalch equation (Equation 2). The pKa was estimated to be 6.4, which is close to that for the rate constant (6.4) (26) or proton uptake (6.6) (27). The two states of PYPM observed at pH 8.5 and at pH 5.0 are hereafter called PYPMalkali PYPMacid, respectively.


Figure 3
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  		FIGURE 3.
Secondary structural change of PYP at various pH values. a, far-UV CD spectra of the PYP dark state (dashed lines) at pH 5.0 and 8.0 and of PYPM (solid lines)atpH 5.0, 6.5, and 8.0. b, difference in molar ellipticity at 222 nm between PYP and PYPM at each pH was plotted against pH.

 
Comparison of Secondary Structure Using CD Spectroscopy—Differences in the secondary structure between PYPMalkali and PYPMacid were evaluated using CD spectra in the far-UV region. The CD spectra of PYP at pH 8.0 and 5.0 agreed with each other in the dark state (Fig. 3a, dashed lines), indicating that the secondary structure of the dark state PYP is pH-independent in this pH region. The CD spectra of the photosteady state were then measured, and pure CD spectra of PYPM were calculated using the fraction of the dark state PYP estimated by absorption spectroscopy under the same irradiation conditions. Typical CD spectra for PYPM at pH values 8.0, 6.5, and 5.0 are shown in Fig. 3a. The significant difference indicates that the secondary structure of PYPMalkali is different from that of PYPMacid. The spectrum at pH 6.5 is in between those at pH 8.0 and 5.0, indicating that it is a 1:1 mixture of PYPMacid and PYPMalkali. The loss of ellipticity at 222 nm using light is 36% for PYPMalkali (pH 8.0) and 9% for PYPMacid (pH 5.0). In Fig. 3b, the light-induced ellipticity change at 222 nm ({Delta}[{theta}]222 nm) is plotted against pH. From pH 8.5 to 5.0, {Delta}[{theta}]222 nm gradually decreased, indicating that the loss of secondary structure is reduced. However, it increased at pH < 5, as demonstrated by the absorption spectroscopy (Fig. 2c), also indicating partial denaturation of PYPM. The apparent pKa for the ellipticity change was 6.4, which agrees with the change in the absorption spectrum. Thus changes in the absorption spectrum of PYPM correspond well with the secondary structural change. Figs. 2c and 3b show that the fraction of PYPMacid is maximal at pH 5.0, and the equilibration shift is almost saturated at pH 8.0. PYPMacid and PYPMalkali were further characterized using PYP solutions at pH 5.0 and pH 8.0, respectively.

Comparison of Secondary Structure Using FTIR Spectroscopy—The difference between PYPMacid and PYPMalkali was studied by FTIR spectroscopy using PYP solution at pH 5.0 or pH 8.0. Spectra were measured in the dark and under continuous illumination, then difference FTIR spectra between photosteady state and dark state were calculated. Vibrational bands between 1500 and 900 cm-1, where chromophore bands appear, were insensitive to pH for the dark state (negative bands); however, the positive bands at 1004 and 1193 cm-1 for PYPMalkali were slightly down-shifted in PYPMacid. This demonstrates a structural difference in the chromophore between PYPMacid and PYPMalkali that would cause an 11-nm difference in the absorption maximum, whereas changes in the protein environment may also contribute.

Global conformational change induced by light is evaluated by the absorbance change in the amide I (~1650 cm-1) and amide II (~1550 cm-1) mode regions. The negative band at 1646 cm-1 at pH 8.0 was reduced and split into two bands at 1644 and 1658 cm-1 at pH 5.0. Second derivative spectra (Fig. 4b) show that a 1646 cm-1 band at pH 8.0 is composed of 1658, 1645, and 1635 cm-1 bands, with the 1645 cm-1 band being the largest of them. At pH 5.0, part of the 1645 cm-1 mode was shifted to 1658 cm-1, resulting in a split peak. A similar shift was observed in N terminus-truncated PYP (23). FTIR spectra and their second derivative spectra clearly show that the 1529 cm-1 band, a typical amide II mode of a beta-structure, was reduced by acidification. Together with the CD spectra results, structural change of both {alpha}-helices and beta-sheets at pH 5.0 is smaller than at pH 8.0. It should be noted that the positive 1747 cm-1 band, which indicates protonation of Glu-46, is observed for PYPMacid. The shoulder of this band suggests that Glu-46 is flexible and in two states in PYPMacid. As the peak area of the negative band at 1736 cm-1 is 5.3 times larger than that of the positive band at 1747 cm-1, it is likely that Glu-46 is partially protonated in PYPMacid.

Solution Structure of PYPMacid and PYPMalkali—The tertiary structures of PYPMalkali and PYPMacid were characterized by small angle x-ray scattering measurements. The scattering profile was measured in the dark and under illumination at pH 8.0 and 5.0. The scattering profiles for PYPM in each preparation (2-10 mg/ml) at pH 8.0 and 5.0 were calculated by subtracting scattering profiles of the dark state from that of the photosteady state, in which the amount of dark state was estimated by UV-visible spectroscopy. Fig. 5 shows Guinier plots (ln I(Q) versus Q2) for dark state PYP at pH 5.0 (a) and 8.0 (b), plus PYPMacid (a) and PYPMalkali (b). All Guinier plots were fitted in the linear region (Q2 < 0.022), and the square of radius of gyration (Rg2) was obtained from the slope. In all samples, illumination of PYP made the slope of Guinier plots steeper, indicating that Rg is increased upon PYPM formation. However, the increase in the slope for PYPMalkali (Fig. 5b) was clearly larger than for PYPMacid (Fig. 5a). Rg2 values estimated at various concentrations were plotted against concentration (Fig. 5c), and the intrinsic values of Rg2 were obtained by extrapolation to a concentration of zero. Rg values thus obtained are shown in Table 1.


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  		TABLE 1
The radius of gyration (Å) of dark state PYP and photoproduct

 


Figure 4
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  		FIGURE 4.
Difference FTIR spectra between PYPM and PYP. a, difference FTIR spectra of dark state (negative signals) and photosteady state (positive signals) produced by 436 nm light. Upper and lower traces were recorded at pH 8.0 and 5.0, respectively. On the lower trace, FTIR spectra at pH 8.0 were superimposed for comparison (dotted line). b, second derivative spectra of FTIR spectra at pH 8.0 (broken line) and pH 5.0 (solid line).

 
At both pH values, y intersections of Guinier plots were not altered by illumination (Fig. 5, a and b), indicating that neither aggregation nor binding of solute ions to PYPM (20) take place. Therefore, the difference in Rg between the samples at pH 8.0 and pH 5.0 simply reflects the difference in the structure between PYPMacid and PYPMalkali. Rg for PYPMalkali (16.2 ± 0.1 Å) was significantly larger than that for PYPMacid (15.7 ± 0.2 Å), indicating that PYPMalkali is swollen compared with the PYPMacid.

Shift in Equilibrium by Citrate—Multivalent organic anions such as citrate bind to PYPM and stabilize it (20). Here the effect of citrate on equilibrium of PYPM was investigated. PYPM spectra at pH 8.0 and 5.0 in the presence of 50 mM citrate were similarly calculated from the photosteady state spectra (Fig. 6a). Both agreed with the absorption spectrum of PYPMalkali at pH 8.0 in the absence of citrate. Under the same conditions, CD spectra of PYPM in the presence of citrate were measured (Fig. 6b). Although CD spectra of PYP in the dark were not affected by citrate, spectra of PYPM at pH 5.0 in the presence of citrate agreed with that for PYPMalkali. In addition, Rg values also agreed with each other (20), indicating there is no difference in protein structure. These results indicate that binding of citrate shifts the equilibrium between PYPMacid and PYPMalkali toward PYPMalkali. In other words, pKa of the equilibrium shifts to less than 5 because of the binding of citrate to PYPM.


Figure 5
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  		FIGURE 5.
Small angle x-ray scattering of dark state PYP and PYPM. Scattering intensity was measured at pH 5.0 (a) and pH 8.0 (b) in the dark (closed circles). It was then measured under continuous illumination and the scattering profile of PYPM calculated (open circles). Data were Guinier-plotted (ln I(Q) versus Q2) and fitted with linear lines. Protein concentrations were 2, 4, 6, and 10 mg/ml (from bottom to top). c, Rg2 was plotted against concentration.

 
The shift in equilibrium caused by the binding of citrate to PYPM was observed by transient spectroscopy at a millisecond time scale (Fig. 7). PYP suspended in 1 M citrate buffer (pH 5.0) was excited by a yellow flash, then transient difference spectra were measured. Just after the flash, an absorbance increase at 360 nm was observed, indicating formation of PYPMacid. It was blue-shifted over time, and finally the positive band of the difference spectra had a maximum at 355 nm and shoulder at 330 nm. This spectral shape agreed with that for PYPMalkali. Absorbance change at 383 nm was plotted against the time after flash (inset), fitted to an exponential curve, and the time constant of this shift was estimated to be 300 ms. In 1 M acetate buffer (pH 5.0), PYPMacid was formed just after the flash, but little spectral shift was observed in this time scale (data not shown).


Figure 6
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  		FIGURE 6.
Shift in equilibrium by citrate. a, absorption spectra of PYPM in the absence of citrate (curve 1 for pH 5.0 and curve 2 for pH 8.0) and in the presence of 50 mM citrate (curve 3 for pH 5.0 and curve 4 for pH 8.0). b, far-UV CD spectra for PYP and PYPM. Curves 1-4 correspond to those in a. Curves 5-8 show CD spectra of dark state PYP in the same buffer system as used for curves 1-4, respectively.

 


Figure 7
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  		FIGURE 7.
Shift of equilibrium by citrate. Dark state PYP in 1 M citrate buffer (base line) was excited by a yellow flash, then transient difference spectra were measured every 40 ms (pH 5.0, 5 °C). Inset, absorbance change at 383 nm was plotted against time after flash and fitted using an exponential curve (k = 0.003 s-1).

 


Figure 8
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  		FIGURE 8.
Equilibrium of E46QM. a, absorption spectra of E46QM at pH 6.5 and 4.0 (solid lines). Absorption spectra of PYPM at pH 2.0, 5.0, and 8.0 are shown for comparison (broken lines). b, difference FTIR spectra between E46Q (negative signals) and E46QM (positive signals) at pH 6.0. PYPM/PYP spectrum at pH 5.0 was reproduced from Fig. 4 (broken line).

 
Role of Glu-46 on Global Conformational Change—Mutation of Glu-46 to Gln results in a significantly reduced conformational change upon the formation of M intermediate (E46QM) (16, 29). Absorption maximum of E46QM at pH 7 is 11-nm red-shifted from PYPMalkali (28), whereas PYPMalkali is dominant at pH 7 for wild type. These characteristics of E46QM are similar to those of PYPMacid. To examine the similarity between PYPMacid and and E46QM, spectroscopic properties of E46QM were compared with PYPMacid (Fig. 8).

Absorption spectrum of E46QM at pH 6.5 was calculated from the absorption spectrum of the photosteady state (Fig. 8a). At pH > 6.5, difference spectra between dark state and photosteady state became significantly smaller because of the short lifetime of E46QM. Absorption spectrum of E46QM at pH 6.5 agreed with that at pH 7.0 obtained from the transient difference spectra 10 ms after the flash (28). At a higher pH, the blue shift of E46QM was not observed, but a significant amount of E46Q was formed (37).4

Absorption spectra of E46QM at pH 6.5 agreed with that of PYPMacid at pH 5.0. At pH 4.0, the spectrum of E46QM was blue-shifted. This agreed with spectra of PYPM at pH 2.0 (acid denatured state of PYPM) and pH 8.0 (PYPMalkali) (Fig. 8a). As it is unlikely that E46QM is converted into its alkaline form by acidification, E46QM is readily denatured at pH 4.0. pKa of phenolic oxygen of the chromophore of E46Q is higher than wild type, suggesting that E46Q is less resistant to acidification than wild type. The apparent pKa of E46QM was estimated to be 5.2 (data not shown).

Difference FTIR spectrum between E46QM and E46Q was recorded at pH 6.0 and then compared with that between PYPMacid and PYP recorded at pH 5.0 (Fig. 8b). The intensity of difference spectra in the amide I and amide II regions shows protein structural change. The shape and intensity of 1608-1575 cm-1 band for E46QM were comparable with those of PYPMacid. PYP at pH 5.0 shows two bands at 1658 and 1644 cm-1. The negative band of E46Q at 1646 cm-1 was not clearly separated, but the shoulder at ~1655 cm-1 would correspond to 1658 cm-1 band of PYP. The negative band of E46Q at 1646 cm-1 is larger than PYP, whereas the positive band of E46QM at 1625 cm-1 was smaller than PYPMacid. Consequently, the difference absorbance between peak (1625 cm-1) and valley (1646 cm-1) for E46Q was 89% of PYP. This indicates that the protein structure of E46QM is comparable with that of PYPMacid. Therefore, absence of negative charge at position 46 would prevent photoactivated PYP from large structural change (16, 29) by shifting the PYPMacid-PYPMalkali equilibrium toward PYPMacid state. In other wavelength regions, overall shape of difference spectra were similar, but significant differences were found. The 1747/1736 cm-1 band of Glu-46 was not observed for E46Q. The 1163 cm-1 and 1302 cm-1 bands of dark state PYP shift to 1158 cm-1 and 1308 cm-1, respectively. The former was observed in the Raman spectroscopy as the shift from 1161 cm-1 to 1154 cm-1 (38), which would mainly cause a red shift of the absorption spectrum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present results clearly demonstrate that the characteristics of PYPM are altered by pH. The pH-dependent changes in absorption spectra and CD spectra were represented with the Henderson-Hasselbalch equation, for both of which the apparent pKa was 6.4. Together with the results of FTIR and SAXS, the pH-dependent change in the nature of PYPM is explained by a pH-dependent equilibrium between two types of PYPM (PYPMalkali and PYPMacid). This is consistent with the previous suggestion by NMR that PYPM (pB) is in an equilibrium between a well ordered state and a partially unfolded state (39). They are different in secondary and tertiary structures as well as the chromophore-protein interaction. The pH-dependent changes in decay rate constant of PYPM (26) and proton uptake (27) are explained by this equilibrium.

The characteristics of E46QM are comparable with that of PYPMacid, suggesting that replacement of Glu-46 by Gln shifts this equilibrium toward PYPMacid. This seems to suggest the protonation of Glu-46 converts PYPMalkali into PYPMacid and vise versa. In fact, a small positive band at 1747 cm-1 was observed in difference FTIR spectrum between and PYPMacid and PYP. This band is assigned to Glu-46 in protonated form, but the peak area of the positive band is 19% of the negative band. As the area is not intensified even at pH 4.0 (data not shown), Glu-46 is not fully protonated in PYPMacid. Therefore it is unlikely that the protonation state of Glu-46 solely switches between PYPMalkali and PYPMacid, although its negative charge is required for a global conformational change (16, 29).

The apparent pKa value of 6.4 in this equilibrium is comparable with that of the imidazole group of the histidine side chain, suggesting the involvement of histidine. The equilibrium shifts toward PYPMacid at low salt concentration (pKa = 6.7, data not shown). As electrostatic interaction is enhanced at low salt concentration, it is likely that PYPMacid is stabilized by the basic amino acid residue, which is charged at acidic pH. PYP has two histidine residues (His-3 and His-108). Although His-3 is fully exposed to the solvent, His-108 is buried between the beta-scaffold and the N-terminal cap. Because the structure of the N-terminal cap changes upon PYPM formation at neutral pH, the protonation state of His-108 possibly correlates with the structure of PYPM (25, 27).

Citrate blue shifts the absorption spectra of PYPM and increases Rg at pH 5 (20). Here the citrate-bound form of PYPM was characterized in detail, demonstrating that the absorption spectrum, the amount of secondary structure and the Rg value of the citrate-bound state of PYPM (PYPM') were in full agreement with those of PYPMalkali. Therefore, the primary effect of binding of citrate is a shift of the equilibrium toward PYPMalkali. NMR analysis of the photoproduct of E46Q has shown that the structural change is limited to {alpha}3 and the chromophore (29). Assuming that the structure of E46Q is comparable with PYPMacid, the main structural change for formation of PYPMacid is the conformational change of {alpha}3. It should be noted that citrate binds to PYPMacid to shift the equilibrium, indicating that the citrate binding site is active in PYPMacid. This finding is consistent with our previous speculation that Arg-52 located in between {alpha}3 and {alpha}4 is flipped and forms the citrate binding site (20).

We previously studied conformational change upon the formation of PYPMalkali by CD spectroscopy (23). Results indicated that the difference maximum in CD spectrum was 222 nm, indicating that a structural change in {alpha}-helices is mainly involved. However, this change cannot be explained by the short {alpha}-helices like {alpha}1 and {alpha}2 located in the N-terminal cap segment. Thus an unfolding of the long helices was strongly suggested. Therefore, the difference between PYPMacid and PYPMalkali is considered to be the unfolding of {alpha}3 and/or {alpha}5. Glu-46 and Thr-50 are located in {alpha}3, and Tyr-42 is located between beta2 and {alpha}3. They are hydrogen-bonded with the phenolic oxygen of the chromophore. It should be noted that Glu-46 is in the central part of {alpha}3. As negative charge at position 46 is required for the conformational change, further structural change in {alpha}3 would take place in addition to that in {alpha}5. FTIR spectra demonstrate that the amide II mode of the beta-sheet (1530 cm-1) is largely changed upon formation of PYPMalkali, indicating that the conformation of the beta-sheet is also changed.

At neutral pH, two types of PYPM (I2/I2' or pB'/pB) are sequentially formed (17, 18). The latter has a lifetime of ~100 ms and is accumulated by steady-state illumination. The former has a lifetime close to PYPL (~1 ms) but the absorption spectrum is close to the latter. The characteristics of pB' are a relatively small protein conformational change (15, 16) and slightly red-shifted absorption spectrum (17). In detailed analysis of pH dependence of the PYP photocycle, data were explained without an equilibrium between pB and pB' (17). However, recent double flash experiments detected a species, which has photoreversal kinetics similar to I2 over ~100-ms time scale, and the authors suggested an equilibrium between I2 and I2' (18). As this experiment was carried out at pH 6, at which 70% of PYPM is in the acidic form, this shows that the photoreversal kinetics of PYPMacid agree with that of I2 (pB'). Absorbance change in amide mode of transient difference FTIR spectrum 450 µs after excitation (15) is comparable with that of the PYPMacid/PYP spectrum. Therefore, it is reasonable to conclude that pB' (I2) and PYPMacid are the same species and that pB' is trapped at acidic pH.

Recently, the crystal structures of photocycle intermediates formed from PYP and E46Q were analyzed by time-resolved crystallography (40-42). They showed the presence of two blue-shifted intermediates of PYP (pB1 and pB2) whose decay time constants are ~10 and ~100 ms (42). These values are consistent with the decay time constants of pB' (PYPMacid, ~1 ms) and pB (PYPMalkali, ~100 ms), respectively. To examine whether or not the structures of pB1 and pB2 correspond to PYPMacid and PYPMalkali, respectively, the SAXS profiles of pB1 and pB2 were calculated from their crystal structures (PDB codes, 1TS0 and 1TS6, respectively) (42) using CRYSOL software (43). Guinier plots of calculated SAXS profile gave the equal values of Rg for pB1 and pB2, indicating that pB1 and pB2 cannot be distinguished by SAXS experiments. Because experimental Rg values for PYPMacid and PYPMalkali are significantly different (Table 1), pB2 would not correspond to PYPMalkali. PYPMacid state in solution would be the mixture of pB1 and pB2, and the large structural change for formation of PYPMalkali is restricted by crystal packing. For E46Q, three late intermediates were found (IL1, IL2, and IL3) (41). IL1 and IL2 show the similar change in electron density observed in PYP. However, no difference in Rg values was resulted from their crystal structures. Together with our results that E46QM is comparable with PYPMacid, blue-shifted intermediates formed in the crystal are qualitatively the same as PYPMacid.

The present data provide detailed characteristics of pB' (I2 or PYPMacid), which is in the stage just before the global conformational change takes place in the photocycle. These observations are of importance in elucidating the mechanism of light-induced global conformational change of PYP.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research (C), for Scientific Research on Priority Areas "Molecular Nano Dynamics" from Ministry of Education, Culture, Sports, Science and Technology and a grant from a Foundation for Nara Institute of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Spring-8/Japan Synchrotron Radiation Research Institute (JASRI), Sayo, Hyogo 679-5198, Japan. Back

2 To whom correspondence should be addressed. Tel.: 81-743-72-6101; Fax: 81-743-72-6109; E-mail: imamoto{at}ms.naist.jp.

3 The abbreviations used are: PYP, photoactive yellow protein from Halorhodospira halophila; SAXS, small-angle x-ray scattering; Rg, radius of gyration; FTIR, Fourier transform infrared; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid. Back

4 M. Harigai, M. Kataoka, and Y. Imamoto, unpublished result. Back


   ACKNOWLEDGMENTS
 
Experiments undertaken at Photon Factory BL-10C were performed under the approval of the Photon Factory Advisory Committee (Proposal numbers 98G191 and 2000G162).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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J. Hendriks and K. J. Hellingwerf
pH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore
J. Biol. Chem., February 20, 2009; 284(8): 5277 - 5288.
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