Energy transfer in allophycocyanin hexamer from Anabaena variabilis by time-resolved spectroscopy

Journal

of

Photochemistry Photobiology and

B:Biology

E LS E V I E R

Journal of Photochemistryand PhotobiologyB: Biology45 ( 1998) 144-149

Energy transfer in allophycocyanin hexamer from Anabaena variabilis by time-resolved spectroscopy Fuli Zhao a,,, Xiguang Zheng a, Jingmin Zhang a, Hezhou Wang a, Zhenxin Yu a, Jingquan Zhao b, Lijin Jiang b "State Key Laboratory of UltrafastLaser Spectroscopy, Zhongshan University, Guangzhou 510275, China bInstitute of Photographic Chemistry, ChineseAcademy of Science, Beijing 100101, China

Received 26 June 1998; accepted 11 September 1998

Abstract The process and mechanism for energy transfer in an allophycocyanin hexamer from Anabaena variabilis have been studied by timeresolved fluorescence spectroscopy. Deconvolution of isotropic time-resolved fluorescence spectra shows that the energy transfer time between two allophycocyanin trimers is about 14 ps, while the energy transfer time from the linker polypeptide (Lcm42) to the final emission chromophore is about 65 ps. The anisotropic results of time-resolved fluorescence spectra show that the energy transfer process among three ring-like (cx-s4/~x-84) chromophore pairs still plays an important role with a time constant of about 45 ps. The facts support the idea that the mechanism for energy transfer in the allophycyanin hexamer should be described by FOster dipole--dipole interaction rather than by an exciton interaction. © 1998 Elsevier Science S.A. All rights reserved. Keywords: Allophycocyaninhexamer; Time-resolvedspectroscopy;Energytransfer; Spectral deconvolution;Excitoninteraction

1. Introduction

2. Materials and methods

Since time-resolved spectroscopy was introduced by Porter et al. [ 1 ] into the research of energy transfer in phycobilisomes (PBSs) and intact algae cells in 1978, this technique has always been the most efficient method to study the process and mechanism of energy transfer in the antenna in algae [2,3]. In the core of the PBS it is difficult to identify the energy transfer paths because high-resolution structural data for the two kinds of allophycocyanin (API and APB) in the intact core are not available and emission peaks of both the isolated proteins are all at about 680 mn. Furthermore, it is more difficult to determine whether the energy transfer rate is controlled by a rate-limiting step or a size-limiting step [4,5]. In this research we have studied the anisotropic fluorescence spectra in detail, discussed the energy transfer paths in the allophycocyanin (APC) hexamer, and compared them with energy transfer processes in the intact phycobilisome.

2.1. Isolation o f A P C hexamer core o f phycobilisome

* Corresponding author. Tel.: +86-20-84197230; E-mail: stszfl@zsv. edv.cn I011-1344/98/$ - see front matter © PII S 1 0 1 1 - 1 3 4 4 ( 9 8 ) 0 0 1 7 3 - 0

(Ot~)5AP~I6.3LcM42from the

The prepared PBSs from Anabaena variabilis were put into a 10 mM K2HPO4-NaH2PO 4 buffer solution (pH = 7.0 containing 1 mM NAN3,2 mM EDTA and 0.5 m M PMSF) at room temperature for 3 h. Then they were applied to a hydroxyapatite column that had been equilibrated with a 10 mM phosphate buffer solution at pH = 7.0. The column was first washed with a 35 mM phosphate buffer solution ( p H = 7 . 0 ) in order to eliminate C-phycocyanin and phycoerythrocyanin. This allowed APCs to remain at the top of the column. Then the column was washed with a 0.75 m M phosphate buffer solution (pH = 7.0). At 20°C, the eluted fraction was immediately applied to a sucrose linear density gradient ultracentrifugation for 16 h at a rate of 43 000 rpm (0.2--0.5 M in 0.75 mM phosphate buffer solution pH = 7.0). The blue band (23S) at the lowest part of the centrifuge tube was the core of the PBS. We applied a 0.05 mM phosphate buffer solution ( p H = 7 . 0 ) to the core preparation at 4°C for 12 h, then we

1998 Elsevier Science S.A. All rights reserved.

F. Zhao et al. / Journal of Photochemistryand PhotobiologyB: Biology 45 (1998) 144-149 Table 1 Spectral data for API and APII

API APII

145

ered the theoretical fluorescence intensity Fth~o as a sum of multi-exponential form:

"~abs

"~max,em

(nm)

(nm)

654 648

680 665

added 0.5 m M / m l et-chymotrypsin with continuous agitation for 30 min; the reaction was stopped by the addition of soya trypsase. At 20°C, the dissociated core of PBS was immediately submitted to sucrose linear density gradient ultracentrifugation (Beckman XL-90, TI-41 ) for 16 h at a rate of 43 000 rpm (0.2-0.5 M in 0.05 mM phosphate buffer solution pH = 7.0). The third band (12S) in the centrifuge tube was the APC hexamer. The characterization of the APC hexamer has been given in detail in a previous paper [6]. It consists of otAP:oLAP:[~I6,3:LcM42in a ratio of 5.1:4.9:1.02:0.98. This implies that the hexamer, isolated by us with this method, in the f o r m o f (O[[~)5AP[~I6.3LcM42 could be regarded as a functional unit of two phycobiliproteins: APII (allophycocyanin II) and API (allophycocyanin I) (Table 1).

Ftheo(t)= )Zei exp(--t/~) We applied a deconvolution procedure based on a global optimization algorithm to fit the isotropic fluorescence. For the anisotropy decay of APC hexamers we have applied the semi-linear Marquardt method to obtain the anisotropic decay r(t): r(t,A) = III(t,A)-g(A)l±(t,A) III(t,k) + 2g(A)l±(t,k) We also applied an iterative Monte Carlo method to search the best result out of the anisotropic fluorescence decays with an error of less than 3.3%. We applied the semi-Marquardt method to find the depolarization time and the function r(t):

r( t,k )= Eeiexp(-t/zi)+ r( O,k ) 3. Results and discussion

3.1. Deconvolution results of steady-state spectroscopy The deconvolution results were as expected (Fig. 1). We assign the shortest-wavelength peak at 600 nm with a large

2.2. Spectral deconvolution 1.0

The steady-state absorption spectra were recorded on a Shimadzu UV-1600 diode array spectrophotometer and the fluorescence excitation spectra were recorded on a Hitachi F-4000 spectrophotometer. All spectra were corrected for instrument response. The deconvolution of steady-state spectra was realized on a computer by non-linear optimization techniques, with the instruction to search the minimum component number. Each band of the component has three parameters: wavelength, peak amplitude and half-width of the band intensity. The sum of all the values of the functions was obtained by estimating the root of the mean square errors.

0.8

8 f-

0.6

¢II 0 w .¢I

0.4

<

0.2

0.0 5OO

2.3. Measurement of picosecond time-resolved fluorescence

1.0

The sample was excited by a cavity-dumped dye laser pulse generated by a mode-locked Nd:YAG laser. The pulse duration was about 6 ps. The fluorescence of the sample was focused into a spectrometer before being collected by a streak camera (Hamamatsu C1587) with a time resolution of 10 ps. The temporal width (fwhm) of the instrument response function (IRF) was less than 10 ps. The fluorescence detected by the streak camera was transferred into an IBM computer for deconvolution. The detected fluorescence intensity F~p can be described as follows:

0.8

Fexp( t)=fpamp®Ftheo= ffpump( t)Ftheo( t - t ' ) d t ' wherefpump represents the pump laser pulse. Here we consid-

700

Wavelength(nm)

~_

0.6 0.4

m u_

0.2

o.o 8OO

65O

7OO

75O

8OO

Wavelength(nm) Fig. l. (a) The deconvolutionof APC hexamerabsorbanceinto four components at 600, 640, 655 and 675 nm, respectively.(b) The deconvolntion of APC hexamerfluorescenceinto four componentsat 640, 660, 680 and 715 nm, respectively.

146

F. Zhao et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 144-149

half-width in the absorption spectra to the absorption of o£84-, ot84-PCB chromophores according to the absorption wavelengths of isolated subunits. The much narrower longwavelength Gaussian bands (640 and 655 nm) seemed to describe the exciton-pair transition Igg > to I + > and t gg > to I - > . The exciton pairs are constructed by adjacent monomers (let84-/21384-PCB, 20t84-/31384-PCB and 3~84-/1884PCB) of each trimer of the APC hexamer, because the centre to centre distance among these chromophores is about 2.08 nm and the coupling interaction between them is about 138 c m - ~ [7]. Unfortunately, the time-resolved emission of such a fast process cannot be detected in our experiment which has a relatively low time resolution. The longest Gaussian band (660 nm) reflects the absorption of the fragment of the anchor polyp•pride ( LCM42). Compared with that of the APC trimer (0t13)3riP, the band of the APC hexamer undergoes a bathochromic shift of 5 nm. This might be caused by the anchor polypeptide (LcM). In the fluorescence deconvolution results (see Fig. l ( b ) ) , the band at 660 nm could describe the APC trimer ( ~ ) 3 AP in this complex, and the band at 680 nm seems to describe the chromophore of the anchor polypeptide fluorescence emission. Based on these results, it can be predicted that the excitation transfer efficiency from APII to API is less than in the intact APC core of PBS.

v 600

800

(nrn)

Fig. 2. Three-dimensional fluorescence spectroscopy of APC hexamer,

•~ex= 570 nm.

3.2. The time-resolved spectral results The time-resolved fluorescence isotropic decays are presented in Figs. 2 and 3. From Fig. 2, we see that there are two fluorescence emission peaks at 660 and 680 nm. The peak at 660 nm disappears quickly in parallel with the increase of the peak at 680 nm, indicating that energy transfer takes place between the two APC trimers. This could be confirmed by the results of Fig. 3 because decay terms at 660 nm corresponded with the rise terms at 680 nm. In order to obtain the fluorescence isotropic decay constants of the APC hexamer, we applied a deconvolution procedure based on a global optimization algorithm. The fluorescence isotropic decay could be well fitted with four exponentials of 14, 66, 130 and 1900 ps, and the amplitudes of each time component are listed in Table 2. The quality of the fit is judged by a global)(2 value of 1.06 (see Fig. 4). These decay constants may be assigned as follows: ( 1 ) The fluorescence isotropic decay constant of 14 ps is due to the energy transfer time between the two APC trimers, via Otsa-PCB chromophores in the APC hexamer, because its amplitude, shown in Fig. 4, is positive at the blue side with a maximum around 650 nm and negative at the red side with a minimum around 670 nm. The negative amplitude represents the fluorescence rise term and indicates that energy transfer occurs from a donor to an acceptor. Therefore, from the fluorescence spectra of the APC hexamer, it is reasonable to assign the decay time of 14 ps to the energy transfer time between the two APC trimers. Sandstr6m et al. [ 8 ] also found the same energy transfer pathway with a time of 16 ps among 18-S complexes that contain a fragment of the APC hexamer.

750 Wavelength

0.8' ~

0.6

ee.

•

•

0.2 o.o

0

,

,

250

,

,

500

,

,

750

,

,

1000

o •o

:

",'~,~

1250

1500

Decay Time (ps)

Fig. 3. Fluorescence intensity decay curves: solid lines, fitting results; Zk, emission at 680 nm; O, emission at 660 nm. A~x= 570 nm.

1.00.8¢1 (Z

0.60.4-

E

o.2-

"1o

0.0-

O~

z

O~./I~N~,ONXX~ '

-0.4-

-0.8~ 18 -

620

S

6;o

do

6;0

7;0

Pml~ Wavelength(nm) Fig. 4. DAS (Decay asociate spectra) of APC fluorescence, ,~ox= 570 nra.

147

F. Zhao et al. / Journal of Photochemistry and PhotobiologyB: Biology 45 (1998) 144-149

Table 2 Deconvolutionresults for isotropic fluorescencein APC hexamer (A~x= 600 nm)

14 ps 66 ps 130 ps 1900 ps

630 nm

640 nm

650 nm

660 nm

670 nm

680 nm

690 nm

700 nm

0.05 0.05 0.05 0.05

0.11 0.18 0.05 0.21

0.19 0.31 0.06 0.58

- 0.18 0.40 0.07 1.00

- 0.80 0.30 0.08 0.70

- 0.61 -0.40 0.07 0.40

- 0.55 -0.20 0.02 0.20

- 0.28 -0.18 0.01 0.05

Data in the first row represent probe wavelengths, while those in the first column represent fluorescencelifetimes (or time components). This is confirmed by our experiment and the analyses are coherent. (2) The fluorescence isotropic decay constant of 66 ps might be assigned to the energy transfer time between chromophores (ct84-, [384-PCB/or [3163-PCB) and the fragment of anchor polypeptide (LcM42).F r o m Fig. 4 we can see that the amplitude for the time constant of 66 ps presents a minimum at 660 nm and a maximum at 680 nm. This can be described as follows: the energy is transferred from the donor whose emission peak is at about 660 nm to the acceptor whose absorption is at about 660 nm and the corresponding emission peak is at 680 nm. Since the structure of API is not available, we could not discuss the donor chromophore in detail from the structural viewpoint. While considering the steady-state spectra of API and the time-resolved fluorescence measurements of both the 18-S complex [ 8 ] and the intact PBS [ 9], we believe that our analyses are reasonable. It should be mentioned that the analysis result of 66 ps in Ref. [9] conflicts with Ref. [8], and our result agrees with Ref. [8]. (3) The fluorescence isotropic decay constant of 1900 ps reflects the final emission of the anchor polypeptide (LcM 42) in the hexamer APC, because its maximum was the same as that of API [ 9 ]. Since the amplitude of the decay constant of 130 ps was less than 8% and it could be submerged within the experimental and fitted error, we did not assign the constant of 130 ps to any energy transfer process. If this constant did originate from a certain energy transfer path, it might be assigned to the energy transfer between 0/.84-and [~s4-PCB chromophores in the same monomer of APC hexamer.

were fitted well by a sum of two exponentials with decay time constants of 11.4 and 37.1 ps, respectively. The shorter time constant of 11.4 is similar to the fluorescence isotropic decay constant of 14 ps within the allowed experimentalerror. Hence, this decay should be assigned to the energy transfer between two different APC trimers via ~s4-PCBs. The decay constant of 37.1 ps, which is not observed in fluorescence isotropic measurements, might be from new energy transfer pathways in the APC trimer (ot[3) 3AP. From the crystal structure of APC [ 11 ], the three [384-PCBs in the centre of the ring-shaped APC trimer (ot13)3 AP, separated by a centre to centre distance of 3.55 nm, are closer together than the ct84-, ot'8n-PfBs chromophores, separated by 6.82 nm. Thus, we predict that the coupling between adjacent [384- and [3'84PCBs should be stronger than that between ot84- and a'84PCBs in APC trimer. The possibility for a new energy transfer process should be between [384--[3'84 pairs, but not between 0t84--(~'84 pairs. As reported by Edington et al. [7], in adjacent monomers of the APC trimer, the 0t84--[~'84 dimers undergo equilibrium rapidly by energy transfer on a femtosecond time

3.3. The results o f time-resolved fluorescence anisotropic decay

~ t )-=0.3016 * (0.24 * e ~ - t/11.4) +0.76 * exp(- t/37.1))+0.1302

0.3

This experiment was carried out to obtain the kinetic information of energy transfer under the consideration of symmetrical effects. This consideration included the energy transfer process between txs4-PCBs in two different trimers and that among three [384-PCBs in the centre o f the ringshaped APC trimer (0L[3)3 AP. The fluorescence anisotropic decays were recorded at 660 and 680 nm with the excitation wavelength at 570 nm [ 10]. The experimental and fitted curves are presented in Figs. 5 and 6. While detecting at 660 nm, we expected that the depolarization kinetic information of the chromophores ct84-PCB and 1384-PCB in APC trimer ( O[1~) 3AP would emerge. The fluorescence anisotropic curves

Panalcl

~

g.

0.2

o.1

""

0.0

~

0

50

'

'

.

.

.

.

.

.

.

~

'

J

'

'

•

J

100 150 200 250 300 350 400 450

Decay Time (ps) Fig.5.Time-resolved fluorescenceanisotropyat660 nm: lines,fittingresults;

scatters, experimentalresults. A~x= 570 nm.

F. Zhao et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 144-149

148

which are formed by the adjacent 1584- and 21384-PCB chromophore pairs in APC trimers. The energy transfer time constant between two APC trimers is about 14 ps via ot84-PCB chromophores in the APC hexamer. The energy transfer time from APC to the anchor polypeptide is about 66 ps and the process is controlled by a rate-limiting step. Additionally, the process of energy transfer among three [~s4-PCB chromophores in either of the APC trimers of the hexamer is still very important with a transfer time of about 40 ps.

Parallel

=/ ~q

o.35

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

5. Abbreviations

(r( t )=0.3237*(O.04*exp(- t/13.6) ) +0.96*exp(- t/40.9 ))+0.1240)

0.30

~"

0.25

PBS APC API APB

~

OA5

Ecru 2 Of.AP

0.10-

o •

0

=

50

.

o i

.

=

.

J

.

=

•

=

.

i

.

/

phycobilisome allophycocyanin allophycocyanin I allophycocyanin B linker polypeptide o~ subunit of allophycocyanin

= .

100 150 200 250 300 350 400 450

DecayTime (ps) Fig. 6. Time-resolved fluorescence anisotropy at 680 nm: lines, fitting results; scatters, experimental results. A~x= 570 nm.

scale, but we cannot detect such a fast process in our experiment which has a time resolution on the picosecond time scale. Therefore it is reasonable to assume that these dimers (exciton pairs) are thermally equilibrated or the exciton energy has been localized when our detection process occurs in the picosecond time range and the dimers can be described as exciton pairs (or as energetically degenerate dimers). As did Debreczeny et al. [ 12] for the C-PC trimer, we could assign the decay constants of 4 0 _ 5 ps to energy transfer among the three energetically degenerate dimers (Ot841 [3842, Ot842~843and 0t843~841) around the trimer ring. When the detection wavelength was selected at 680 nm, similar fluorescence time components were obtained. The two time constants are 13.6 and 40.9 ps, and are the same as those identified at 660 nm. Thus we conclude that the 40.9 ps process occurs with three ring-like chromophore pairs, and the final emission chromophore (or donor) is the anchor p o l y p e p t i d e Lcm 42, which acts as the trap of the APC hexamer.

4. Conclusions With picosecond time-resolved spectroscopy and the application of deconvolution techniques, we have studied the energy transfer paths, energy transfer time constants and the mechanism of energy transfer in the APC hexamer. The results indicate the following: in the hexamer the components of the absorption spectrum at 640 and 655 nm may come from the transitions [gg > ---, [ + > and ]gg > --" I - > ,

Acknowledgements This work has been supported by the National Scientific Fund of China (NSFC), grant No. 19574077; we gratefully acknowledge Ms Judith A. Coopy for careful reading and modifying the manuscript thoroughly.

References [ 1 ] G. Porter, C.J. Tredwell, G.F.W. Searle, J. Barber, Picosecond timeresolved energy transfer in Porphyridium cruentum, Part I, Biochim. Biophys. Acta 501 (1978) 232-245. [2] A'R" H°lzwarth' Structure-fnncti°n relati°nships and energy transfer in phycobiliprotein antennae, Physiol. Plant 83 ( 1991 ) 518-528. [3] R.S. Knox, D. Gitlen, Theory of polarized fluorescence from molecular pairs: Ffrster transfer at large electronic coupling, Photochem. Photobiol. 57 (1993) 40-43. [4] D J. Lundell, A.N. Glazer, Molecular architecture of a light-harvesting antenna: allophycocyanin assembly in the core substructure of the Synechococcus 6301 phycobilisome, J. Biol. Chem. 256 (1983) 12600-12606. [5] P. Fuglistaller, M. Mimuro, F. Suter, H. Zuber, AUophycocyanin complexes of the phycobilisome from M. laminpsus: influence of the linker polypeptide Lc89 on the spectral properties of the phycobiliprotein subunits, Biol. Chem. 368 (1987) 353-367. [6] J.M. Zhang, J. Xie, J.P. Zhang, J.Q. Zhao, L.J. Jiang, Isolation and characterization of allophycocyanin hexamer from A. variabilis, Acta. Biophys. 13 ,, 1997) 173-178 (in Chinese). [7] M.D. Edington, R.E. Riter, W.F. Beck, Evidence for coherent energy transfer in allophycocyanin trimer, J. Phys. Chem. 99 (1995) 1569915704; Interexciton-state relaxation and exciton localization in APC trimer, J. Phys. Chem. 100 (1996) 14260-14267. [8] A. Sandstr6m, T. Gillbro, V. Sundstr6m, J. Wendler, A.R. Holzwarth, Picosecond study of energy transfer within 18-S particles of A N 112 (a mutant of Synechococcus 6301 ) phycobilisomes, Biochim. Biophys. Acta 933 (1988) 54--64. [9] A.N. Xia, J.C. Zhu, L.J. Jiang, X.Y. Zhang, Energy transfer process among PBS from Wesiellopsisprolifica at 77 K, Science in China (B) 25 (1995) 277-282.

F. Zhao et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 144-149 [ 10] A.A. Demidov, D.L. Andrews, Determination of fluorescence polarization and absorption anisotropy in molecular complexes having threefold rotational s y ~ , Photochem. Photobiol. 63 (1996) 39-52. [ 11 ] K. Brejc, R. Ficner, H. Huber, Isolation, crystallization, crystal structure analysis and refinement of allophycocyanin from the cyanobacterium Spirulirm platensis at 2.3 /k resolution, J. Mol. Biol. 249 (1995) 42 A, A.A.9.

149

[12] M.P. Debreczeny, K. Sauer, J. Zhou, D.A. Bryant, Comparison of calculated and experimentally resolved rate constants for excitation energy transfer in C-Phycocyanin. Part I: monomers, J. Phys. Chem. 99 (1995) 8412-8419; Part II: trimers, J. Phys. Chem. 99 (1995) 8420-8431; T. Schirmer, W. Bode, H. Hnber, Refined three-dimensional structure of two cyanobacterial C-phyeocyanins at 2.1 .~ and 2.5/~ resolution, J. Mol. Biol., 196 (1987) 677-695.