Photosynth Res DOI 10.1007/s11120-015-0166-1
REGULAR PAPER
Heat-induced unfolding of apo-CP43 studied by fluorescence spectroscopy and CD spectroscopy Qing-Jie Xiao1 • Zai-Geng Li1 • Jiao Yang1 • Qing He1 Lei Xi1 • Lin-Fang Du1
•
Received: 21 January 2015 / Accepted: 3 June 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract CP43 is a chlorophyll-binding protein, which acts as a conduit for the excitation energy transfer. The thermal stability of apo-CP43 was studied by intrinsic fluorescence, exogenous ANS fluorescence, and circular dichroism spectroscopy. Under heat treatment, the structure of apo-CP43 changed and existed transition state occurred between 56 and 62 °C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.10 ± 1.02 °C by calculating a-helix transition and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C by calculating the unfolded fraction of tryptophan and tyrosine fluorescence, respectively. During the process of unfolding, the hydrophobic structure of C-terminal segment firstly started to expose at 40 °C, and then the hydrophobic cluster adjacent to the N-terminal segment also gradually exposed to hydrophilic environment with increasing temperature. Our results indicated that heat treatment, especially above 40 °C, has an important impact on the structural stability of apo-CP43. Keywords apo-CP43 Thermal stability Fluorescence ANS Circular dichroism Structural rearrangement Abbreviations RC Reaction center EET Excitation energy transfer CD Circular dichroism & Lin-Fang Du
[email protected] 1
Key Laboratory of Bio-resources and Eco-environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, People’s Republic of China
IPTG PSII RLS IS kex kem kmax F350 BSA ANS MRE
Isopropyl thio-b-D galactoside Photosystem II Resonance light scattering The intermediate state Excitation wavelength Emission wavelength Maximum emission wavelength Fluorescence intensity at 350 nm Bovine serum albumin 8-anilino-1-naphthalenesulfonic acid Mean residue ellipticity
Introduction The photosystem II (PSII) complex is a large supramolecular pigment protein complex embedded in the thylakoid membranes of green plants, algae, and cyanobacteria (Hankamer et al. 1997). The central part of PSII is the reaction center (RC), where the charge separation and water splitting occur. The RC is surrounded by the outer antenna protein. CP43 (inner antenna protein) is located in the reaction center and acts as a conduit for excitation energy transfer (EET) from the outer antenna of PSII to the reaction center (RC) (Bricker and Frankel 2002). Besides, it can also play an important role in maintaining the structural integrity and the oxygenevolving capacity of PSII (Zouni et al. 2001; Loll et al. 2005; Umena et al. 2011). In contrast to the outer antenna protein, CP43 is relatively conserved among all types of oxygenic organisms (Boekema et al. 1995). It contains six transmembrane a-helices, which are separated by five extrinsic loop domains and binds 13 Chls a and 3 b-Cars (Zouni et al. 2001; Loll et al. 2005; Umena et al. 2011). In the spinach,
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apo-CP43 contains 473 amino acid residues, and the relative molecular mass is about 52 kDa (Alfonso et al. 1994). Previous studies indicated that the deletion of the psbC gene resulted in a loss of photoautotrophic growth and oxygen-evolving capacity (Vermaas et al. 1988; Rochaix et al. 1989; Barber et al. 2000) and has a profound effect on the assembly of functional PS2 core complexes in vivo (Rochaix et al. 1989; Shimada et al. 2008). The research showed that CP43 is connected with 33 kDa protein by the largest hydrophilic loop (Enami et al. 1997) and had a close association with Psb27 (Liu et al. 2011). Besides, it also provided an amino acid ligand to the Mn4Ca cluster (Ferreira et al. 2004). Interaction among pigment proteins is related to the apoprotein. In addition, with the improvement in the resolution of the crystal structures of PS II complexes (Zouni et al. 2001; Bricker and Frankel 2002; Loll et al. 2005; Umena et al. 2011), the energy transfer mechanism has become one of the focuses of recent studies, and the pigment plays a key role in energy transfer (Mu¨h et al. 2012). However, apo-CP43 provided the rack for the pigment binding, so its structure largely affected the energy transfer among pigments. So far, the structural and functional properties of apo-CP43 still remain an open question. In the study, we focused on the heat treatment-induced functional and structural aspects of apo-CP43 by means of spectroscopic assays, such as intrinsic fluorescence, exogenous ANS fluorescence, and far-UV circular dichroism (CD) spectroscopy. These researches will pave the way for the role of pigment in maintaining the CP43 structure, and the studies of CP43 reconstitute in vitro the energy transfer among pigments and so on.
Materials and methods Materials BL21 E. coli strains were used for expression and purification of the apo-CP43. HisTrap HP prepacked with NiSepharose High Performance was obtained from Amersham. Tris, SDS, ANS, and Isopropyl thio-b-D galactoside (IPTG) were purchased from Sigma Chemical Company (USA).
were incubated at 27 °C for 4 h. The cells were pelleted by centrifugation at 4000 rpm for 20 min at 4 °C and then resuspended in buffer A (20 mM Tris–HCl, 10 mM imidazole, and 500 mM NaCl, pH 7.5–8.0). The cells were lysed by ultrasonic oscillator for 20 min. After centrifugation at 10,500 rpm for 40 min, the supernatant was purified by Ni-chelating column in the AKTA FPLC system (Liu et al. 2014a). The column was washed using buffer A until the UV reading dropped down to about 0. The apo-CP43 was directly washed by buffer B (20 mM Tris–HCl, 500 mM imidazole, and 500 mM NaCl, pH 7.5–8.0). The protein (0.7 mL) was further purified by Sephacryl S-100 HR and washed with Tris–HCl (20 mM pH 7.5) (Liu et al. 2014b). SDS-PAGE analysis was used to detect the purity. In addition, the protein concentration was determined according to the improved Lorry assay method using bovine serum albumin (BSA) as a standard. Measurements of the intrinsic fluorescence spectra Fluorescence measurements were performed on a Hitachi F-4500 fluorescence spectrophotometer equipped with a circulating water bath as described before (Wang et al. 2012). Samples were prepared in a quartz cuvette with 1 cm path length. Excitation and emission slit widths were set at 5 nm and 10 nm, respectively. Typically, apo-CP43 in 20 mM Tris–HCl (pH 7.5) was incubated from 20 to 95 °C for 10 min before the fluorescence spectra were recorded. The intrinsic tryptophan fluorescence was measured between 310 and 400 nm with the excitation wavelength of 295 nm. The synchronous fluorescence spectra were recorded between 250 and 350 nm with Dk (constant wavelength interval, Dk = kem – kex) of 15 or 60 nm (Mu et al. 2011). Every outcome was the mean of at least three scans. Because of the absorption of the exciting light and the reabsorption of the emitted light, the fluorescence intensities were corrected to decrease the inner filter effect as described before (Lakowicz and Masters 2008; Ding et al. 2010): Eq. (1): Fcor ¼ Fobs eðAex þAAem Þ=2 ; where Fcor and Fobs are, respectively, the corrected and observed fluorescence intensities and Aex and Aem are, respectively, the absorbance of the solution at the excitation and emission wavelengths. Calculate the fraction of unfolded apo-CP43
Expression and purification of apo-CP43 The gene of apo-CP43 was cloned into the PET-28a expression vector, and E. coli BL21 harboring the recombinant plasmid was cultured in 500 ml medium containing kanamycin (30 lg/ml) at 37 °C. When the OD600 reached about 0.6, IPTG (1 mM) was added into the cultures, which
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The fluorescence intensity at each temperature was normalized by deducting the disturbance from the thermal quenching (Yamamoto et al. 2006; Wang et al. 2010a). The fraction of unfolded apo-CP43 at each temperature was calculated using the following equation (Pace 1986; Wang et al. 2010b):
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FD ¼ ðFt F0 Þ=ðFd F0 Þ;
ð1Þ
where FD is the amount of unfolded apo-CP43 at a temperature, Ft is the fluorescence intensity at the temperature, and F0 and Fd are the fluorescence intensities of the native and denatured proteins, respectively. Tm is the temperature 50 % of apo-CP43 being unfolded and can be calculated by plotting FD against temperature, by fitting the data to sigmoidal curves using Origin 8.0 software. Measurements of the ANS fluorescence spectra ANS is an extrinsic fluorescence probe that binds to the hydrophobic cluster of protein (Paul et al. 2008). The fluorescence spectra of ANS (50 lM) were investigated upon incubation with apo-CP43 (2.5 lM) at different temperatures for 10 min (Busby et al. 1981; Wicker et al. 1986; Ding et al. 2010; Wang et al. 2012). After cooling to room temperature, the spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer. Excitation and emission slit widths were, respectively, set at 5 nm and 10 nm for ANS fluorescence spectra. The ANS fluorescence spectra were recorded in the range of 400–600 nm with kex = 380 nm (Wang et al. 2012; Zhang et al. 2011). Measurements of circular dichroism spectra The far-UV CD measurements were carried out on an AVIV Model 400 circular dichroism spectrophotometer as described before (Wang et al. 2013). Apo-CP43 (2.5 lM) in 20 mM Tris–HCl (pH 7.0) was incubated from 20 to 90 °C for 10 min before the spectra were recorded. Signals were collected from 200 to 260 nm in a quartz cuvette with 1 mm path length. Each outcome was the mean of at least three scans.
Results and discussion Change in the intrinsic fluorescence spectra of apo-CP43 induced by heat treatment The fluorescence emission spectra provide information about the microenvironment of the Tyr and Try residues, which can be used as an indicator of structural changes. The intrinsic fluorescence is sensitive to the tertiary structure and the conformation of protein in solution. In the process of protein unfolding, the polarity of their surrounding environment gradually increases which leads to the change of fluorescence emission spectra (Kuma et al. 2005; Lakowicz and Masters 2008). The native CP43 in spinach contains 17 tryptophan residues and 15 tyrosine
residues, with six tryptophan and five tyrosine residues located in the transmembrane helices (Bricker and Frankel 2002; Anderson et al. 2002). Figure 1 shows the changes in the fluorescence spectra of apo-CP43 with the increasing temperature. At 20 °C, the highest fluorescence intensity was observed and the kmax was located at 340.6 nm with kex = 295 (Fig. 1A), suggesting that the most tryptophan residues were located in relatively hydrophobic environments in apo-CP43 (Lakowicz and Masters 2008; Esquembre et al. 2013; Drake and Hoops 2014). However, the kmax is not obviously shifted with the elevated temperature. In order to investigate if temperature can induce changes in the microenvironments of the tryptophan and tyrosine residues, the F350/F330 ratio (Fig. 1B) was used to monitor the shifts in the kmax of the fluorescence emission spectra (kex = 295 nm) (Wang et al. 2011). In Fig. 1B, the shift of F350/F330 ratio was small from 20 to 45 °C, indicating that the hydrophobicity of the microenvironments around the tryptophan residues slightly changed. The ratio increased obviously from 45 to 53 °C, but was a tiny change from 53 to 59 °C that possibly means state transition of apo-CP43 after obvious change of the microenvironments around tryptophan residues (Wang et al. 2011; Drake and Hoops 2014). With the temperature increase, the F350/F330 ratios gradually increased which showed that the polarity in the microenvironments of the tryptophan residues increased further (Pace 1986; Samuel et al. 2000). In addition, the synchronous fluorescence spectra with the Dk = 60 can also present some information about the microenvironment of the tryptophan residues (Lakowicz and Masters 2008; Wang et al. 2013) shown in Fig. 1C. The fluorescence quenching occurred with the temperature increase; however, the kmax is not obviously shifted similar to the fluorescence spectra with kex = 295. When analyzing the F295/F280 ratio in Fig. 1D, the change of the F295/ F280 was apparent from 40 to 53 °C, but was small from 53 to 59 °C and from 62 to 68 °C. However, there existed an obvious change between 59 and 62 °C indicating the change of apo-CP43 state (Samuel et al. 2000). The fluorescence emission spectra with kex = 295 nm and the synchronous fluorescence spectra with the Dk = 60 mainly provided the microenvironment change of the tryptophan residues in apo-CP43. On the other hand, the synchronous fluorescence spectra with the Dk = 15 can present some information about the tyrosine (Lakowicz and Masters 2008; Bobone et al. 2014). The synchronous fluorescence spectra (Dk = 15 nm) of apo-CP43 with the increasing temperature are plotted in Fig. 2A. The highest fluorescence intensity was observed at 20 °C, and the kmax was located at 288.6 nm. The fluorescence quenching occurred with the elevated temperature and the kmax shifted from 288.6 to 287.2 nm. In order to further investigate the change in the microenvironment of tyrosine,
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Photosynth Res Fig. 1 Thermal-induced structural changes in apo-CP43 as monitored by the intrinsic fluorescence emission spectroscopy. The fluorescence emission spectra of apo-CP43 with kex = 295 (A) and structural changes in the F350/ F330 ratio for the spectra with kex = 295 (B). The F295/F280 ratio for the spectra Dk = 60 (C, D). a–h 25, 35, 45, 56, 65, 75, 85, and 95 °C
Fig. 2 Thermal-induced structural changes in apo-CP43 as monitored by synchronous fluorescence spectroscopy (Dk = 15). A The synchronous fluorescence spectra of apoCP43 with Dk = 15. B Structural changes in the F295/ F280 ratio for the spectra with Dk = 15. a–h 25, 35, 45, 56, 65, 75, 85, and 95 °C
the F280/F295 ratio of the spectra is shown in Fig. 2B. The F280/F295 ratios gradually increased from 20 to 53 °C, but there was a small change from 53 to 59 °C and from 62 to 68 °C. There existed obviously a transformation between 59 and 62 °C suggesting state transition of apo-CP43 after heatinduced structural change (Samuel et al. 2000). The ratio increases sharply from 68 to 71 °C and then slowly with the increasing temperature. The synchronous fluorescence spectra (Dk = 15) indicated that there existed the change in the hydrophobicity around the tyrosine residues and it is gradually increasing with the elevated temperature (Samuel
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et al. 2000; Esquembre et al. 2013; Drake and Hoops 2014). These results above indicated that high-temperature treatment disrupted the substructures around both of the tryptophan and tyrosine residues in apo-CP43, especially above 40 °C, and the heat-induced structural changes were different in the polarity and hydrophobicity around tryptophan and tyrosine residues. Besides, there was a state transition occurred between 59 to 62 °C which is further stated below. Furthermore, we calculated the fraction of unfolded apoCP43 using Eq. (1) based on the fluorescence intensities of the tryptophan and tyrosine residues and plotted the
Photosynth Res Fig. 3 Fraction of unfolded apo-CP43 monitored by the changes in fluorescence intensity. A Fraction of unfolded apo-CP43 calculated based on the fluorescence emission intensity at kex = 295 nm. B Fraction of unfolded apo-CP43 calculated based on the synchronous fluorescence intensity at kex = 293 nm
normalized data in Fig. 3A, B. Apo-CP43 gradually unfolded, when the temperature was above 40 °C. The unfolding process was monitored by tryptophan fluorescence (Fig. 3A) and tyrosine fluorescence (Fig. 3B) and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C, respectively. The unfolding process slowed down from 56 to 59 °C which confirmed the existence of state transition as shown in Fig. 3. The further discussion is stated below. Temperature-induced conformational transition in apo-CP43 revealed by ANS The inner fluorescence spectroscopy only indicates the microenvironments of the tryptophan and tyrosine residues, so more methods should be applied to perform a systematic investigation of the thermal unfolding of apo-CP43. ANS is a much-utilized probe for examining exposure of the hydrophobic clusters. The fluorescence of free ANS is very weak in aqueous solution. However, once bound to the hydrophobic clusters of protein, the fluorescence intensity of ANS increases significantly and kmax of the spectra will shift toward the blue light spectrum (Pace 1986; Gasymov and Glasgow 2007; Paul et al. 2008). ANS and apo-CP43 together were incubated at different temperatures for 10 min. It is different from the process that apo-CP43 was firstly treated at different temperatures and then ANS was added. In the process of apo-CP43 refolding, ANS gradually binds to the hydrophobic segment of apo-CP43. Therefore, ANS fluorescence intensity is closely related to the degree of apo-CP43 unfolding (Wicker et al. 1986; Shi et al. 1994; Ding et al. 2010; Wang et al. 2011). Figure 4A shows the changes in the ANS fluorescence spectra of apoCP43 with the increasing temperature. At 20 °C, the lowest fluorescence intensity was observed and the signal was enhanced with the elevated temperature. In order to further investigate the change, the maximum fluorescence intensity and F490/F510 ratio were used to assess the change as shown in Fig. 4B. The change was relatively small from 20
to 40 °C. However, the ANS fluorescence intensity obviously increased to about 68 % and the F490/F510 ratio shifted from 0.925 to 0.976, when the temperature is increased from 40 to 60 °C, indicating the unfolding of apo-CP43 and the binding of ANS to the hydrophobic surface (Gasymov and Glasgow 2007; Paul et al. 2008). However, the change was slow, and there appeared a reduced trend between 60 and 70 °C and then obviously reduced above 70 °C indicating the existence of state transition. The reduction of ANS fluorescence is possibly due to the aggregation of apo-CP43 leading to the concealment of partial hydrophobic cluster. These results above showed heat-induced denaturation of apo-CP43 started at 40 °C, and the exposure of hydrophobic clusters increased with the elevated temperature. In order to further explore the structural change in apoCP43, hydrophobicity of apo-CP43 was analyzed by the DNAman software, as shown in Fig. 5. The results showed that the hydrophobicity of apo-CP43 obviously differed between the two segments from the 1 to 291 amino acid residues that contain 5 transmembrane a-helices and from the 291 to 473 amino acid residues that contain 1 transmembrane a-helix and the most hydrophilic loop, respectively (Bricker and Frankel 2002). Due to the obvious difference in the two segments, the sensibility to the heat can differ. The fluorescence spectrum showed that possibly there existed a state transition between 56 and 62 °C after heat-induced change in apo-CP43. Besides, exposure of hydrophobic cluster started at 40 °C due to the change of ANS fluorescence spectrum and there existed a state transition at about 60 °C. Based on these results, it can be inferred that the heat induce the unfolding of N-terminal and C-terminal segment existing difference. The segment adjacent to the C-terminal firstly unfolds, due to the relatively weak hydrophobicity. However, unfolding of the segment adjacent to the N-terminal may need relatively higher temperature due to the stronger hydrophobicity.
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Photosynth Res Fig. 4 Heat-induced unfolding of the hydrophobic pocket in apo-CP43, as revealed by ANS fluorescence spectroscopy (kex = 380). A The fluorescence spectra of the apoCP43-ANS system. B Changes in the maximum fluorescence intensity and in the F490/F510 ratio
Fig. 5 The prediction of hydrophobicity of apo-CP43
Temperature-induced conformational transition in apo-CP43 revealed by CD spectroscopy In order to further reveal the alteration in apo-CP43 after the heat treatment, the far-ultraviolet CD spectrum is used to assess quantitatively the overall secondary structure content of the protein (Greenfield 1996; Kelly and Price 1997; Kelly et al. 2005). The CD spectrum in the farultraviolet region (200–260 nm) for apo-CP43 showed two negative peaks, which changed with the elevated temperature, as shown in Fig. 6. The CD spectrum of apo-CP43 at room temperature (Fig. 6A line a) was similar to that of the native CP43 purified from spinach (Li et al. 1997) or that given by another research group (Wang et al. 1999) showing that the primary structure plays an important role to structure of CP43. However, the second negative peak of apo-CP43 is relatively steady compared with that of native CP43, indicating that the pigments have some influence on the structure of apo-CP43. The CD activity in the far-ultraviolet region gradually decreased during heat treatment. We further plotted the band intensity at 208 nm that is related to helix (Greenfield 1996), as shown in Fig. 6B. With the elevated temperature, the band intensity at 208 nm gradually increased, especially between 40 and
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70 °C. In order to further investigate the change of structure with the increasing temperature, the secondary structure element of apo-CP43 was analyzed using Cdpro software (Pace 1986) as shown in Fig. 6C, and the changes of a-helix were shown in Fig. 6D. The content of a-helix gradually decreased with the elevated temperature, especially above 40 °C, and the isosbestic point of the sigmoidal curve was 58.1 ± 1.02 °C that was close to the Tm which was observed by the fluorescence experiments. In addition, content of b-sheet gradually increased with the temperature, especially above 50 °C. The possible reason was due to the unfolding of apo-CP43 that leads to the formation of extra intermolecular b-sheet (Kato et al. 2005; Qu et al. 2007; Wang et al. 2012; Naeem et al. 2011).
Conclusion The present study described the measurement of the heatinduced unfolding of apo-CP43, monitored by intrinsic fluorescence, exogenous ANS fluorescence, and far-UV CD spectroscopy. These results showed that the change of apoCP43 structure by the heat treatment mainly occurred at above 40 °C, and there existed a transition state between 56
Photosynth Res Fig. 6 Far-UV CD spectra of apo-CP43 in the buffer at temperature from 30 to 80 °C. A Far-UV circular dichroism spectra. B Temperature-induced changes in the band intensity at 208 nm. C Secondary structure of apo-CP43 at various temperatures. D Plotting a-helix against the temperature. a–f 30, 40, 50, 60, 70, and 80 °C
and 62 °C that is close to the thermal transition of native CP43 occurred at 59 °C which was proposed by another research team (Qu et al. 2007). The analysis of hydrophobicity of the apo-CP43 showed an obvious distinction between the two segments adjacent to the N-terminal and C-terminal, respectively, so the sensitivity to heat can have some difference. According to these results, the thermal unfolding of apo-CP43 can include two periods, ranging from 40 to 60 °C and from 60 to 70 °C. The hydrophobic structure of C-terminal segment firstly started to unfold at 40 °C, and then the spatial structure and hydrophobic cluster mainly located in the N-terminal segment were further exposed with the elevated temperature. Wang et al. 1999 and Qu et al. 2007 detected heat to affect CP43 structure by the CD technology, Fourier transform infrared spectroscopy, and terahertz time-domain spectroscopy. The result showed two temperatures of state transition that occured at 50 and 59 °C, respectively. Therefore, it could imply that heat may affect apo-CP43 which was a two-state process. In addition, some clues can be acquired by the unfolding process of apo-CP43, especially the substructures around the tryptophan and the tyrosine residues. The unfolding process monitored by tryptophan fluorescence and tyrosine fluorescence revealed that the Tm is about 56 °C which was similar to the isosbestic point of a-helix content that
was 58.1 ± 1.02 °C. Result of Far-UV CD spectra shows the higher content of alpha-helix compared with the other secondary element. Therefore, the alpha-helix was used to calculate the isosbestic point that can reflect the heat to affect the apo-CP43 secondary structure. However, the intrinsic fluorescence spectra of one protein is sensitive to the tertiary structure and the conformational stability of it in solution (Kazakov et al. 2009; Wang et al. 2010a). Altogether, it contributes to the more comprehensive analysis of the thermal effect on the structure of apo-CP43 and explores the relationships between secondary and tertiary structures. Moreover, the CD spectrum of apo-CP43 is similar to that of the purified native CP43 in the far-UV region (Wang et al. 1999). The result above showed that the primary structure is largely related to the structure and stability of CP43, but there existed some distinction due to that the pigments have some role in sustaining the structure (Wang et al. 1999; Liu et al. 2014a). These results obtained will be helpful in understanding the role of pigment in apoCP43 and the experiment will be continued to further reveal the mechanism of induced conformational transition. Acknowledgments This work was supported by the National Basic Research 973 Program 2009CB118502, the National Natural Sciences Foundation of China (No.30870181, 31170223), and the Doctoral Foundation of Ministry of Education of China (20070610168).
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