Modelling of the photosystem II 33 kDa protein: Structure, functions and possible sulfate-sensitive sites derived from sequence-encoded information

hmi~o,mtotla/,rod l;x/,oim*l~ta! B,,la/¢t V . [ :Q, N o {. p p I l l

[2~. 1992

l'.im, d m (;,(:~l lJlitain.

0098 8472,92 S51)tl I tk00 I 1992 Pll~illtlqlll PILES [Ad

M O D E L L I N G OF THE P H O T O S Y S T E M II 33 kDa PROTEIN: S T R U C T U R E , F U N C T I O N S A N D POSSIBLE S U L F A T E - S E N S I T I V E SITES D E R I V E D F R O M S E Q U E N C E - E N C O D E D I N F O R M A T I O N MARC BEAUREGARD

Chenfistry Department, University of Prince Edx~ard Island, 555 University Avenue, (:harlottetown, P.E.I., Canada (:IA 4P3

(Received 3 April 1992: acc@led ip~ revi~ed,/brm 3 ,]u!~, 1992) BEAI'REGARDl~l. Alodelli*zg q/'lhe Pholos_l~alo*z11 33 kDa proleiH: .~lruclure, funclions and po.~sible ,~ulfillesa~ri/ive ,~iles dericed,/?om .~equence-em'oded i,!/brmalion. E N V I R O N M E N T A L A N D E X P E R I M E N T A L B O T A N Y 32, 411 423, 1992. A recent study revealed that SO., inhibits higher plant Photosystem II (PSlI) activity via the action of sulfate on two oxygen-evolving complex ['unctions: (I) the binding of chloride, and (2) the binding of the 23 and 18 kDa extrinsic proteins (BEAUREGARD, M. (1991) Envir. exp. Bol. 31, 11 21). The "33 kDa" protein of PSII is involved in these txxo [ttnctinllS and theretbre reprcs('nts a key to thc understanding of sulthtc action at the molccular level. This protcn is, however, poorly characterized, regarding both structure and functions. Hme the inti)rmalion content of the 33 kl)a protein froin seven species is analyzed and used to build a working model tot this protein. l'hc prediction presented corroborates slructural data available in the lileraturc tbr spinach 33 kDa protein. Three fl-strands (around L146, $202 and (;215}, an exposed a-helix at Q185 and many chain revcrsals were predicted tbr lbur 33 kDa proteins from higher plants. A similar consensus lbr secondary structures and exposure propensit3 is tl,und tbr cyanobacterial proteins, whereas the ion-binding sites proposed for higher plants are not well cnnservcd in c) anol)acteria. For higher plants: (1) a (alcium-/)inding site is proposed around KI01, (2) seven domains ¢t"~l to G2, Q12 to TIS, Y45 to K49, K101 to EI(/3, K137 to 1) 139, R178 to E182 and D207 to E209) and 13 residues are identified as possible stflfatc-sensitix e sites in spinach 33 kDa protein. The results arc integrated in the first model [br the 33 kl)a extrinsic protein of Phntosvstem [ 1 that hlcludes structure and functhms.

Key ~e,0r&: Sulflu" dioxide, oxygen-evolving conlplcx, sequence analysis, secondary structure )redicti(m, inn-billding site, )ollutant.

INTRODUCTION

O E C protein necessary fbr tull oxygen-evolving activity in plants, :'° and (2) neither the 23 nor "FHE acUvitv o[" the Photosystem II (PSII) oxythe 18 kDa proteins are present in cyanobacteria, gen-evolving complex (OEC) in the higher plant where tire 33 kDa c o m p o n e n t is sufficient to allow stable oxygen evolution in vivo. 47 M a n y fhnctions involves three extrinsic proteins located on the h | m e n a l side of the thylakoid, designated 33, 23 }rave been proposed for the 33 kDa protein, and 18 kDa (apparent molecular masses estinamely the stabilization of manganese atoms (hereafter referred to as M n , the oxidation state mated by polyacrylamide gel electrophoresis). ':~7':;!~ A m o n g these, the 33 kDa polypeptide being variable) involved in water splitting; 4e the has the most i m p o r t a n t role: (1 it is the only acceleration of the transition $3 to S. in the S41 1

412

M. BEAUREGARD

state cycle; TM the provision of binding sites for the 23 and 18 kDa proteins, ~31'37i except in cyanobacteria; <47 and the inclusion of binding sites tbr calcium and chloride ions. l:~,~0,54, It has been demonstrated that PSII OEC is sensitive to sulthte present as a stable product of SO 2 translbrmation in plant cells under realistic levels of atmospheric SO2. ~l It is known that sulfate inhibits OEC by two modes of action: (a) competition tbr chloride's binding sites; and (b) interference with electrostatic attractions that bind the 23 and 18 kDa subunits to the PSII complex.' i, The 33 kDa protein, which is believed to bear sites for chloride binding and which is necessary for the binding of the 23 and 18 kDa subunits, :~7' is theretbre central to the understanding ofsull~tte inhibition of higher plant PSI I. The next step in determining the mechanism of sulthte inhibition of PSII OEC is the identification ofsull~ate targets in the 33 kDa protein. Because the importance of the 33 kDa protein tbr PSII efficiency is generally recognized, bacterial expression systems have been designed to allow easy purification, investigation and engineering of this proteinJ 44 However, the potential of this powerful approach is limited by the absence of a model tbr 33 kDa protein that would include structure and active sites. Today, not much is known about the 33 kDa protein at the molecular level and the fb.w insights provided so f~tr regarding its structure do not allow construction of a testable model. The main elements that have been reported about 33 kDa protein structure are: its amino acid sequence (about 250 residues);~< its approximate dimensions, proposed to be similar to a disk of 7 nm diameter and 1.53.3 nm thickness; c-''-' and the presence ofa disulfur bridge between C28 and C51, which appears necessary tbr the binding of" the 33 kDa protein to PSI1/:<4!'' An important step toward the establishment of a testable model that will allow identification of the sensitive sites in the 33 kDa protein is an analysis of its amino acid sequence. Previous proposals for 33 kDa protein active sites 1'~'4~'did not take advantage of this source of inlbrmation. Conserved properties, such as segment hydrophylicity, chain flexibility, and residue exposure propensity, help in locating the potentially active sites. Also, the sequence itself contains structural

inlbrmation that can be revealed by applying recently developed structure prediction methods. It is now generally recognized that the amino acid sequence holds all the information required tbr most proteins to achieve proper folding, aS' Although long range interactions cannot be predicted and used to draw tertiary fblding, shortrange interactions between neighbouring residues have allowed successful prediction or matching of the secondary structures of many proteinsJ 5'j°':~:< Success in modelling natural protein TM and in de n0v0 protein design ~2 further supports the potential of secondary structure prediction methods. The object of this contribution is to gain insights into the 33 kDa protein structure, and to suggest domains and amino acid residues that should be determinant tbr its functions and sensitivity to sult~te. For this, the amino acid sequences of 33 kDa protein from tbur higher plants and three cyanobacteria are compared and analyzed. The results of this study allow the construction of a model that provides testable predictions and lays the foundation for engineering of the 33 kDa protein via site-directed mutagenesis. MATERIALS AND M E T H O D S

Sequence al~gnmenl The sequences of 33 kDa protein ti'om Anabaena sp. PCC 7120, ~' ~nechococcus sp. PCC 7942, '~ Syneehocys& sp. PCC 6803, ~< spinach, ~"~ pea, < wheat 3G~ and potato ~ were aligned using Clustal, a multiple alignment program developed by HIOGINSand SHARV.''2>

Periodic secondar~ ,~lructureprediclion Location of ~-helices and fl-sheets was pertbrmed here using a combination of three complimentary prediction methods (Homologue, G O R III and Bit-pattern) as described previously.-' Hoinologue's predictions are based on the structural similarity which exists among short homologous peptides. ~:~4 G O R III uses intbrmation theory, namely the information carried by 17 neighbouring residues in a segment about the secondary structure assumed by the residue at the centre of this segment. 2~ The bitpattern method considers the distribution of hydrophobie/hydrophilic residues along the sequence, given that specific distributions are

M O D E L L I N G OF THE PSII 33 kDa PROTEIN observed in 0~-helices and in f l - s h e e t s f '2!~' T h e h y d r o p a t h y scale used is derived from the solvent exposure of the 20 amino acids in 41 proteins with X-ray resolved structure. :5 T h e first two methods have a higher weight in the design algorithm, as detailed in BIOtT el a l l '~ Elements of more than five consecutive residues with preti'rence tbr c~helix were retained, while tbr fl-sheets a m i n i m u m of tbur residues was required tbr assignment, unless otherwise specified. W i t h these criteria, a level of 75~'{, correct prediction tbr regular secondary structure runs was o b t a i n e d when the method was tested with a 67 p o l y p e p t i d e d a t a base. 5

Prediclion oJchain reversal (loop) Given that chain reversals contain exposed residues i m p o r t a n t lot activity and a t t a c h m e n t / recognition of proteins, a prediction p a t t e r n was [bllowed which was i n d e p e n d e n t of the secondary structure prediction described above. First, the propensity of a given residue to be in a buried or exposed segment was estimated. For this, the h y d r o p h y l i c i t y of residues was calculated along the sequence using the approach of KYTE and DOOtJTTI.E, with a window of seven residues. '~:~ Both w a t e r - v a p o u r transt~r free energies and protein interior exterior distribution of a m i n o acid side-chains are considered in the h y d r o p a t h y scale. :!' Calculated m a x i m a l / m i n i m a l values showed good correlation with exposed/buried portions of g l o b u l a r proteins. ~:~ Second, a n o t h e r estimation of exposure preterence of a m i n o acid residues based on calculation of surt:ace p r o b a b i l i t y 17 was also perlbrmed. For this, the empirical a m i n o acid accessible surface p r o b a b i l i t y d e t e r m i n e d ti'om structural d a t a of 28 proteins e7 was used. T h e h y d r o p h y l i c i t y and surlhce prol)ability m i n i m a and m a x i m a along the sequence were c o m p a r e d . Residues predicted to I)e exposed have a h y d r o p h y l i c i t y p a r a m e t e r higher than 1.2, and a surtitce p r o b a b i l i t y of more than 1.5. T h e threshold values tbr buried residues were set t() - 0.8 and 0.7, respectivelx. T h i r d , the p()ssible locations of fi-turns were then assigned using c o m m o n prediction iiom two methods. First, the empirical m e t h o d of CHOU and FaSMAN, which simply relies on tile propensity of a given residue to be in a given structure, was used. ii,: T h e p r o b a b i l i t y of at fi-turn is

413

based on the frequency at the first, second, third and tburth positions in turns of 29 characterized proteins, and assignments are done with the procedure described by NISHIKAWA. 4°! Second, the G O R method was a p p l i e d without o p t i m i z a t i o n tot turn prediction. ~> O n l y the residues as turns by both methods were predicted as possible fiturn elements. Fourth, tile flexibility m a x i m a were localized using the method of KARPLUS and NCHULZ. :~0 Residues with an index of 1.05 and more were assumed to indicate a flexible segment. T h e predictions were then considered together to d e t e r m i n e the most p r o b a b l e loop residues. ( 1) O n l y the charged or exposed (as defined above) residues were considered. (2) From these sites, only those that are also predicted as fi-turns, or as flexibilit} m a x i m a , or that are proline, were retained. (3) Finally, the predicted loop residues that were located within a predicted ~-helix, flsheet or buried locus were discarded. [In thct virtually no conllict was found between loop and periodic structure prediction. O n l y one loopprone segment was tbund to overlap with a periodic structure element (e-helix No. 1 in Anabaena, see Fig. 1)]. T h e residues r e m a i n i n g were then considered as indicative of a chain reversal element (loop) that should include other neighbouring residues. This loop prediction method is essentially based on criteria developed by COH~:.~, ,\BARBANEL, KUNTZ a n d FLETTERICK. II

RESULTS AND DISCUSSION

T h e relative amounts ot" ~-helix and fl-strand of the seven 33 kDa protein a m i n o acid sequences !see Methods) are s u m m a r i z e d in T a b l e 1. F r o m information on tile expected accuracy of the combined a p p r o a c h used hcre (750. correct location of secondary structures, see Methods), it can be concluded that the ti-aetion of a m i n o acids in periodic structures is lower than 3 0 ° . in 33 kDa protein. The average inter-loop numher ot'residues is 13, a value close lo that observed tor other proteins with a similar a r r a n g e m e n t o[':~ and fl structures. :11 In ~theat, ,tnabaena, and spinach, the :¢-helix residues dominate. This is at variance with Svnectzoc>li.~, &,nechococcus and pea. However, the general correlation between secondary strutlure ot'spinach nnd .lnabaena, for example, is not

414

M. BEAUREGARD

Table 1. Predicted percenlagea cf secondarr slruclure in 33 kDa proleim

Periodic structure (~}b) Species

~-helix

fi-shcet

dlmhaena 3'rnechococcu.~ S~,lwchocy.~li~ Pea Spinach Wheat Potato

14 7 3 3 13 21 12

6 10 13 12 9 9 12

Average:

10 (3)

(3) (3) (1) (1) (3) (7) (4)

Average inter-loop length (residues)

(4) (6) (7) (6) (4) (4) (6)

15 17 14 11 10 12 10

10 (5)

13

The values arc given in percentage of residues in the specified structure. Values in parentheses refi'.r to the number of structured segments. Averages lot the seven proteins are given at the bottom. better than tbr other pairs, as shown in Fig. 1, where structural predictions tor indivicual proteins are given. The structure elements were slightly more conserved in higher plants (or in cyanobacteria) when compared separately, as expected. Prediclion vs experimenlal J'acls The amount of c~-helix residues in spinach 33 kl)a can be obtained finm near-u.v. CD spectra reported previously by TANAKA and WADA. 19 Using the value of" ka~2~ ti'om the spectra to calculate the ~-helix percentage, a we calculate that about 9~!i, of the residues adopt an c~-helix contbrmation in spinach 33 kDa protein. In view of these data, the prediction done here fbr the c~helix content of spinach 33 kDa protein is quite satislactnry (Table 1). The prediction shown in Fig. 1 tbr spinach is also in agreement with the recent results of a digestion study of spinach 33 kDa protein.'~c' The. stability of secondary structures upon digestion of the first 18 residues suggests that no periodic secondary structure is present at the N-terminal in spinach, in agreement with the structure prediction presented in Fig. 1. Funclional delerminanls Cyanobacterial 33 kl)a proteins are different fi'nm higher plant proteins in that they do not

bind 23 and 18 kDa proteins, '.7 the proteins that regulate the calcium and chloride requirement for higher plant O9-evolution. :~°'2<39~ In spite of this, it has been shown that the 33 kDa protein from cyanobacteria and spinach could be interchanged to partially reactive oxygen evolution activity in PSII fi'om other species. ~:~>Therelbre, the identification of the conserved characteristics in the seven proteins should provide a meaningl'ul way of locating important domains in the 33 kDa protein. In Fig. 2 are shown the conserved properties in both cyanobacteria and higher plant proteins. Since higher plant 33 kDa proteins have about 85~Io homoh)gy, wheat and potato are not shown in this comparison to avoid redundancy. For structure, only the tbatures conserved in five sequences or more ate shown, which reveals that most sequence-encoded inff)rmation ahout structure is c()nserved only in the C-terminal half of the sequence: many loops near G43, K66, K105, E 121, K 137, then a fi-strand conserved from F 145 to V147, three other loops near R151, K159 and D180, tbllowed by an exposed ~-helix around K186, then a buried fl-sheet fi'om T200 to T204, a loop at K207, tbllowed by another fi-sheet from V213 to F217 and finally a loop region including $223 and D224. Note that the residues (and their number) are taken from the spinach sequence which serves as the reference sequence throughout the text. The prediction of a loop between the two adjacent buried fi-strands suggests that these strands are antiparallel and stabilize each other. The consensus lot exposure a m o n g the sequences is also limited to the C-terminal half" of the 33 kDa proteins. The conservation of properties in the 33 kDa proteins was not well correlated to residue homology. For example, the segment 180 to 218 containing the conserved ahelix and two/]-strands has only 20'~ o conserved residues against an overall 31~i, homology, tbr the five proteins shown in Fig. 2. The structural homology revealed by sequence-encoded intbrmarion could obviously not be detected by looking at residue homology. T h e conservation of these structures suggests that they m a y be of vital importance for 33 kDa f'unction and reveal a high level of" homology a m o n g the different species. At the N-terminal, it has been proposed that the segment L6 to I l l and t h e S S bridge (C28,

MODELLIN(; OF THE PSII 33 kDa PROTEIN

Anabaena Synechococcus Synechocystis Pea Spinach

Anabaena Synechococcus Synechocystis Pea Spinach

1

1 Ii ii

--al--

415 1

1

1

iii 1 Ill--B1-1 1 iii 1 1 iii 1 1 1 . . . . al . . . . EGG-K-RLTYDEIQSKTYLEVKGTGTANQCPTV-EGGVDSFAFKPGK-YTAKKFCLEPTK i0 20 30 40 50

. . . . . . . . . ~i . . . . . iiiiiiiii iii iiii 1 1 1 -~i-ii 1

1 -BI-

1

ii

1 -B21

ii 1 --BI-II iii 1 1 1 iii

-B2-BI-

FAVKAEGISKNSGPDFQNTKLMTRLTYTLDEIEGPFEVSSDGTVKFEEKDGIDYAAVTVQ 60 70 80 90 i00 ii0

Anabaena Synechococcus Synechocystis Pea Spinach

1 1 1 1 1 1

-B2-B3-

1 1 -B4ii 1 1

LPGGERVPFLFTIKQLVAS-GKPE 120 130

Anabaena Synechococcus Synechocystis Pea Spinach

-B4-

1 iii 1 ii--~2. . . . . . ~i . . . . i i i i iii---~I--iii iiii--~2--iii

ii ii 1 - B 3 - 1 1 1 1 -B3I-B2-

1 ii 1 1 1

1 -B21 1 1 1 1 ii 1 1

........ SFSGDFLVPSYRGSSFLDPKGRGGSTGY 140 150 160 ~2 ..... B31 -B51 ---B6-Ill--B4-1 ---B3--

1 1 iiii iii

--B4-1111111 --B6ii --B7iiiii --B5ii ---B4--iii

1 1 1

DNAVALPAGGRGDEEELQKENNKNVASSKGTITLSVTSSKPETGEVIGVFQSLQPSDTDL 170 180 190 200 210 220

Anabaena Synechococcus Synechocystis Pea Spinach

1 ---~3----a3-Ii --B6ii . . . . ~3 . . . . . . GAKVPKDVKIEGVWYAQLE--QQ 230 240

lhG. 1. (1omparison of individual secondary structure predictions tbr the 33 kDa proteins. Symbols arc: ( 1 ; loop, '.m) ~-helix, (fl) /J-strand. The sequence is given tot spinach 33 kDa protein. The alignment is tim samc as in Fig. 2.

(15l) p a r t i c i p a t e in b i n d i n g 33 k D a to P S I I . 1~.4~, F r o m the comparison of the structural properties predicted fbr the segment L6 to 111 tbr the seven sequences, it a p p e a r s that some ligatures are poorly conserved (Fig. 1, and d a t a not shown). For example, L6 is predicted to be buried in ,~),ne-

chococctls, whereas the same residue is exposed in ,~nechocyslis and spinach. No p a r t i c u l a r secondary structures are conserved a m o n g the proteins in this region, but residue homology within this segment is very high. T h e sequence p a t t e r n L6, T7, ( g or F)8, (D or E)9, (Q, E or D) 10, and I l l is

Anabaena Synechococcus Synechocystis Pea Spinach

ASSTRDILTYEQI--RG ..... ATGLANKCPQLTETSRGSIPLDSSKSYVLKELCLEPTN TAADLGTLTYDEIQIKD ...... TGLANKCLSLKESARGTIPLEAGKKYALTDLCLEPQE FAVDKSQLTYDDI-VN ....... TGLANVCPEISSFTRGTIEVEPNTKYFVSDFCMEPQE EGAPK-RLTFDEIQSKTYLEVKGTGTANQCPTI-DGGVDSFSFKPGK-YNAKKLCLEPTS i0 20 30 40 50 EGG-K-RLTYDEIQSKTYLEVKGTGTANQCPTV-EGGVDSFAFKPGK-YTAKKFCLEPTK

homology structure exposure charges

Anabaena Synechococcus Synechocystis Pea Spinach

**..

Pea Spinach

**

**

*

. . . . . . . . . .

*

...*.**

FFVKEEPANKRQTAEFVAGKLLTRYTSTIDQVSGDLKFNDDSSLTFVEKDGLDFQAITVQ FFVKEEPGNKRQKAEFVPGKVLTRYTSSLDQVYGDLALKADGTVSFTEKGGIDFQAITVL YFVKEEPVNKRQKAEYVKGKVLTRQTTSLEQIRGSIAVGADGTLTFKEKDGIDFQPITVL FTVKSEGVTKNTPLAFQNTKLMTRLTYTLDEIEGPFEVSADGSVKFEEKDGIDYAAVTVQ 60 70 80 90 i00 ii0 FAVKAEGISKNSGPDFQNTKLMTRLTYTLDEIEGPFEVSSDGTVKFEEKDGIDYAAVTVQ **.*

+

.* . . . . 1 e +

-

*..**

+

*

.....

+

*

.* .... * * * . * . * .... * * 1 bbbb -+ -

-

LPGGERVPFLFTIKNLVAQT-QPGLSSLNTSTDFEGTFKVPSYRGSAFLDPKGRGVVSGY LPGGEEVPFLFTVKGLVASTSEPA-TSINTSTDLRGGYRVPSYRTSNFLDPKARGLTTGY LPGGEEVPFFFTVKNF-TGTTEPGFTSINSSTDFVGDFNVPSYRGAGFLDPKARGLYTGY LPRGERVPFLFTIKQLVAS-GKPD ........ SFSGEFLVPSYRGSSFLDPKGRGASTGY 120 130 140 150 160 LPGGERVPFLFTIKQLVAS-GKPE ........ SFSGDFLVPSYRGSSFLDPKGRGGSTGY

homology structure exposure charges

**

Anabaena Synechococcus Synechocystis

DNAVALPAQA--SSEDLTRTNVKRAEILNGKISLQIAKVDSSSGEIAGTFESEQPSDTDL ESAVAIPSAG--DAEDLTKENVKRFVTGQGEISLAVSKVDGATGEVAGVFTAIQPSDTDM DNAVASPSA .... ADKF-RTNKKETPLGKGTLSLQVTQVDGSTGEIAGIFESEQPSDTDL DNAVALPAGGRGDEEELGKENNKSAASSKGKVTLSVTQTKPETGEVIGVFESIQPSDTDL 170 180 190 200 210 220 DNAVALPAGGRGDEEELQKENNKNVASSKGTITLSVTSSKPETGEVIGVFQSLQPSDTDL

Pea Spinach homology structure exposure charges

.

1

homology structure exposure charges

Anabaena Synechococcus Synechocystis

*

**

***

**

*

.

.*.

i

..

*..

1

*****...*****.**

SSB

1

.**

i

bbbbbbb -

..***

+

.. . . . . . . 1

*

_

Spinach homology structure exposure charges

.....

BBBBB bbb

+

Anabaena Synechococcus Synechocystis Pea

.*...*

.

uu~u eee ee

bbb _

- +

+

**...*

1

+

BSB~B bbb -

+

******. ii ee _

_

GADEPKEVKIRGIFYARVE .... GGKEAVDVKLVGQFYGRIEPADA GAKEPLDVKVRGIFYGRVD-TDV -AKAPKDVKIQGVWYARLN--HR 230 240 GAKVPKDVKIEGVWYAQLE--QQ .

.

.

.

**.

*

.*

. . . .

Fro. 2. Compilation of'conserved properties in 33 kDa proteins. Residues conserved are marked with " * " , and conservative mutations are indicated by ".". The five species compared in this particular alignment are ,lnabaena sp. PC(-: 7120, @nechococcussp. 7942, Synechoc~stis sp. 6803, spinach and pea. Secondary structure predictions (a: a-helix, [;~:/~-strand, 1: loop), and exposure propensity (b: buried, e: exposed) are given when |ouud in five proteins out of seven (wheal and potato are not shown; see text). The spinach sequence is numbered arid used as the retbrence sequence throughout the text.

MODELLING OF THE PSII 33 kDa PR()TEIN conserved in all proteins studied here and also in tobacco.= 1~. This suggests that an epitope involved in binding of the 33 kDa protein to PSII overlaps with this segment. A similar rationale prevails for tile S S bridge between C28 and (151 tbund in spinach. '~' These two residues are conserved ill all proteins studied here. The comparison of structure prediction, locus characteristics and residue homology indicates that the bridge demarcates a protein segment without conserved properties (secondary structure and exposure are ditt'erent). However, the formation of the S-S bridge brings together two segments of high residue homology. This observation suggests that once brought together by S-S bridge formation, the segments "1'22 to (128 and (151 to P54 tbrm a continuous, well conserved seginent that could be involved ill 33 kI)a tunctions and binding in both cyanobacteria and higher plants (Fig. 2). A negative charge is conserved in the segment 1,6 to I11 (D9) and a second one in the segment C51 to P54
Binding sties./or 23 and 18 kDa proleins Tile binding of tile 23 and 18 kDa proteins to PSII is sensitive to anions including sulthte. <3~

tl7

The interactions that link these proteins involve segments ti-oln tile 33 kDa protein. ::~7":~l We can thus expect targets ofsulthte action on the 33 kl)a protein. Up to now, no proposal has been made tbr these sites. We can identi~: possible binding sites tbr the 23 and 18 kDa proteins on higher plant 33 kDa protein by taking advantage of the absence of binding sites on cyanol)acterial 33 kl)a tbr these proteins. ~7 Exclusive homology among 33 kDa proteins tiom higher plants is evidenced bv the li~w gaps in tile alignment indicated in Fig. 2. In addition, the structural characteristics, charges and residues exclusively conserved in higher phmt 33 kDa proteins were considered to lind extrinsic protein-binding sties. Many properties are conserved exclusively in higher planls, especially a large number of exposed [ n - 191 and charged [n = 171 residues absent ti'om cyanobacteria (Fig. 3}. The participatilm of charged residues in binding is expected ti~om the known properties of 23 and 18 kl)a protein binding. Both polypeptides itre released its pH or sah content increases in solution. 4.:~s~ Theretbre residues contributing to electrostatic attraction and/or residues undergoing pH induced change in the range 6 8 are potential binding sites. The only residues expected to change charge in the pH range are histidine and cvsteine. No such residues in 33 kl)a protein which can be involved in tile pH-induced release of the 23 and 18 kDa proteins are identified in Fig. 3. Such residues are, however, available in the 23 and 18 kDa protein, e~ Electrostatic attractions involving seven charged and exposed domains around El, K14, K50, K101, K137, El80 and K207 in spinach 33 kDa could pardcipate in the binding of the 23 and 18 kDa protein. The domains provide 11 charges, more than required tbr binding two proteins. ~;

Cation-binding sties Three types of ion-binding sites were proposed in plant 33 kDa protein: Mn-, Ca ''~-, and chloride-binding sites. In cyanobacteria, the situation is difl'erent. A recent study ruled out any binding of Mn to 33 kDa protein.' j: Moreover, the calcium and chloride effect/role was observed ill higher plants and is highly affected by the 23 and 18 kDa proteins, both absent in cyanobacteria. The segment El8 to G33 in spinach (Fig. 2) was pro-

418

M. BEAUREGARD

posed as the Mn-binding site due to its similarity with bacterial Mn superoxide dismutase ( M N SOD). ~l' Out of 19 residues involved in such Mn-binding sites of M N - S O D , only 10 can be matched with a conserved segment of wheat, pea, spinach and potato 33 kDa proteins, c o l responding to El8 (}33 in Fig. 2. A poorer homology between this MN SO1) Mn-binding site and the corresponding segment in ChlamMomonas reinhardlii was also mentioned, c~5 In cyanobacterial 33 kDa proteins, only five residues match the bacterial Mn-binding segment. For all proteins, the Mn ligand in this site should be C28, according to the alignment. <: The S-S bridge fbrmation between C28 and C51 ~l~ sheds doubts on this proposal. This and the titct that cyanobacterial 33 kDa (that does not bind to Mn in cyanobacteria has low homology to the Mn-binding site) can reactivate O2-evolution in higher plants indicate that 33 kDa protein has a rather indirect impact on Mn functions. Various proposals have been made fbr possible calcium-binding sites in 33 kDa protein. PHILBRICK and ZILINSKAS 43' proposed the region INSSTDF in 5)'nechoeyslis, a segment absent in higher plants (aligned with El40 in spinach). COLEMAN and GOVINDJEETM considered most E and D residues ignoring other putative calcium ligands- in spinach protein as potent calciumbinding sites. Among these sites, the residues El, 18, 103, 139, 209 and D36, 144, 180 (in spinach) are not conserved in cyanobacteria. These proposals suffer fiom a lack of similarity with known calcium-binding sites; further, ira calcium-binding site exists in cyanobacteria, one would expect this segment to be more or less conserved in higher plants. In this line of reasoning, another proposal tbr a calcium-binding site in 33 kDa protein is based on the high homology found between the segment T84 to T114 of pea 33 kDa protein and the calcium-binding domain (EF-hand type) of intestinal calcium-binding protein (ICaBP). ~5'~ We thrther tested this proposal by comparing the 33 kDa protein sequences to the EF-hand consensus sequence obtained fbr four types of calcium-binding protein. (4s The comparison (not shown) reveals that 33 kDa proteins fi'om fbur higher plants have a very high residue homology Io the EF-hand sequence. However, a glutamate (E93) is fbund at the first ligand position, where

an invariant aspartate is found for EF-hand. :4~ The EF-hand have a typical structure (helix loop-helix) that is not predicted for any of the 33 kDa proteins. The accuracy of the prediction done here was tested on ICaBP and the helixloop-helix domain binding calcium was correctly assigned ( > 90(}i, of residues). The recent claim ~4: that 33 kDa protein would have "all structural requirements" for the tbrmation of an EF-hand is contradicted by the study presented here. In tact, no available structural fbrmation suggests the presence of a conserved helix loop-helix domain in 33 kDa protein. Moreover, the rather low content of~-helices predicted tbr tile 33 kDa protein is at variance with the high c~-helix content of many characterized calcium-binding proteins. 4~< A calcium-binding site that is not flanked by c~helices has been [bund in periplasmic D-galactosebinding protein (GBP). :52i The segment has an insert of 62 residues at position 10 of a typical EFhand site but is highly homologous to the segment 93 to 104 in spinach 33 kDa protein. The prediction of structure in 33 kDa protein is compatible with such a calcium-binding site in higher plant 33 kDa protein. Also, slight diff?rences between the 33 kDa protein-binding site and the well known EF-hand may be explained by the effect of the 23 kDa protein on the calcium requirement tbr O.,-evolution. '-'1'4~'Some segment ti~om 23 kDa protein could complement a partial calciumbinding site in 33 kDa protein; we have seen above (Fig. 3) that an extrinsic protein-binding domain is predicted in the same region. The homology between cyanobacterial proteins and the calcium-binding site is not as good, with tbur to five residues in conflict with the 12 residue consensus sequence. 4~' There are no 23 or 18 kDa proteins in cyanobacteria to complement an incomplete Ca 2*-binding site. Thus, it can be concluded that cyanobacterial 33 kDa proteins have no Ca 2 ~-binding site homologous to known sites, while higher plants could have a site around K101.

Anion-binding .riles The chloride-binding sites are the other type of target tbr sulfate. COLEMAN and GOVINDJEF,'l:~'~ have already discussed possible chloride-binding sites in spinach 33 kDa protein. Salt bridges were

M O D E l , L I N G O F T H E PSII 33 kDa P R O T E I N Pea Spinach homology structure charge exposure flexibility binding

Pea Spinach homology structure charge exposure flexibility binding

Pea Spinach homology structure charge exposure flexibility binding

Pea Spinach homology structure charge exposure flexibility binding

Pea Spinach homology structure charge exposure flexibility binding

41

EGAPKRLTFDEIQSKTYLEVKGTGTANQCPTIDGGVDSFSFKPGKYNAKKLCLEPTSFTV i0 20 30 40 50 EGG-KRLTYDEIQSKTYLEVKGTGTANQCPTVEGGVDSFAFKPGKYTAKKFCLEPTKFAV **

*

-

+

iii

1

+

--

+

+

ee e

e

eee

~

KSEGVTKNTPLAFQNTKLMTRLTYTLDEIEGPFEVSADGSVKFEEKDGIDYAAVTVQLPR 60 70 80 90 i00 ii0 KAEGISKNSGPDFQNTKLMTRLTYTLDEIEGPFEVSSDGTVKFEEKDGIDYAAVTVQLPG

e

GERVPFLFTIKQLVASGKPDSFSGEFLVPSYRGSSFLDPKGRGASTGYDNAVALPAGGRG 120 130 140 150 160 170 GERVPFLFTIKQLVASGKPESFSGDFLVPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRG 1 +

ii

--

+

e

e

e

e

DEEELGKENNKSAASSKGKVTLSVTQTKPETGEVIGVFESIQPSDTDL-AKAPKDVKIQG 180 190 200 210 220 230

DEEELQKENNI~IVASSKGTITLSVTSSKPETGEVIGVFQSLQPSDTDLG/~'VPKDVKIEG ii ee

ii

ee

VWYARLNHR 240 VWYAQLEQQ

ee

e

FI(;. 3. Compilation of propcrties exclusively conserved in higher plant 33 kDa proteins (absent from the three cyanobactcrial 33 kDa proteins). Peptide segments proposed to participate in binding the 23 and 18 kl)a proteins are identified by " ~ ". Symbols are as in Figs l and 2. The consensus is not modified when potato and wheat sequences are included.

420

M. BEAUREGARD

proposed to explain the observation that chloride binding apparently requires protonation of a group with a p K a of" 5. '~5' Such a group would be salt-bridged to potential anion sites (arginine, lysine), leaving the sites available at p H > 5. Once bound, chloride could be released by an increase o f p H over 8. ~4' The evidence presented tbr such a concept is discredited by the etti~ct of p H on 23 and 18 kDa protein binding, which was not considered in these studies. Obviously the pHdependent release of the 23 and 18 kDa proteins, :4~ i.e. the polypeptides controlling the chloride requirement of oxygen-evolution by PSII, drastically limits the value of such a proposal. Theretbre, no such bridges are proposed here. Rather, the emphasis is laid on conserved potential anion-binding sites. T h e properties of the C1 effect in PSII were compared to those of various C1 -sensitive enzymes. '~> It was concluded that the most likely C1 -binding sites in the 33 kDa protein were basic residues, not metal centers. 1'2) Positively charged residues at p H values encountered in the thylakoid lumen (i.e. about 5 7) are lysine, arginine and histidine.

-

A modelfor the 33 kDa protein oJPSH Finally, the results were integrated in the model shown in Fig. 4. Although the model is intended

++

V~-~

IO0

M a n y of these are conserved in the seven 33 kDa proteins, and correspond to K60, K66, K76, R80, K105, K I 3 0 , R151, K159, R161, K 1 8 6 a n d K190 in spinach (Fig. 2). When the higher plants (wheat, potato, pea and spinach) were compared separately, 13 additional conserved sites were tbund, namely K4, R5, K14, K20, K41, K44, K48, K49, K101, R122, K137, R178 and K233 (Fig. 3). Higher plant 33 kDa proteins have nearly twice as m a n y conserved positively charged residues as the general consensus. Once again, the ion sensitivity is probably different for 33 kDa protein o f c y a n o b a c t e r i a as c o m p a r e d to higher plants. A m o n g the candidates, only those that are freely accessible to other anions are expected to be chloride-binding sites. (26 T h e exposure propensity which is sequenced-encoded was studied here (Figs 2 and 3). It narrows the list of potential anion-binding sites down to 13 residues: K14, K48, K49, K66, K101, K105, K137, K159, R161, R178, K186, K190, K233.

(D

--

YYYrY'(~Y-~ 10

90--

--

+

+ 20

+

7O ~

It1

I s

30

S

40 N~ +

I

+

-- + 60

+

110 _ ~

__.~

170 -

(])- ' ~ ~ J ' ~ J ~

14o -

190

_(2o 230

--

240

C

FIG. 4. Model fbr the 33 kDa protein of PSII OEC in higher plants. Spinach is used as the template. Symbols are defined as: (@) for anion-sensitive and ( ~ ) for extrinsic protein binding sites, i.e. both sulfate-sensitive sites; ( ~ ) exposed region; (---) buried locus; (c',,), (,u) loops; ( ~ ) s-helix; (,,,w) fl -strand; ( y ) PSII-binding segments; and ( + / - ) for conserved charges. The disulfur bridge between C28 and C51 and the putative Ca2+-binding site are indicated. All elements of tile model were conserved in the four higher plant 33 kDa proteins. See text for details.

MODELLING OF THE PSII 33 kDa PROTEIN tbr higher plant 33 kDa, some of its elements are also valid tbr cyanobacteria. These elements are (1) the secondary structures: three fl-strands, an exposed ~-helix, and m a n y chain reversals; (2) the exposed and buried regions; and (3) the residues involved in b i n d i n g the 33 kDa protein to PSII. T h e c o m m o n a l i t y of these three ligatures was inw~lved when it was tbund that a cyanohacterial 33 kl)a could be exchanged with a spinach protein to obtain partial PSII activity. ~'-' T h e poor homology fimnd at the level ot" i o n - b i n d i n g sites tor cyanobacteria explains at least in part why this exchangeability gave only partial reactiwttion. l:or higher plants, a c a l c i u m - h i n d i n g site is suggested a r o u n d K I 0 1 , [3 residues are proposed to hind chloride and seven domains arc candidales tot h i n d i n g the 23 and 18 kDa proteins. T h e sullhte-sensitive sites are to be tbund a m o n g the chloride- and p r o t e i n - b i n d i n g sites. In this regard, some residues K14, K48, K49, K101, K 137 and R 178 have both capacities. T h e moditication of their e n v i r o n m e n t by protein engineering could increase the chh)ride specificity b} preventing the approach of bigger anions like sullhte. This next step provides a way of testing the accuracy of this model of 33 kl)a protein aim opens the way to rational modification of PSII sensitivity to sulfate.

4.

5.

6.

7.

8.

9.

10.

1l. Acknowledgment At the time this research was done, the author xxas supported by a postdoctoral [~llowship [i'om the Natural Sciences and Engineering Research Council of Canada. The author would like to thank Dr R. M. Teather tbr critical reading of the manuscript.

12.

13. REFERENCES 1. BEAUR~:GARDM. ( 1991 ) Involvement ofsulfite and sulfhte anions in the SO2-induced inhibition of the oxygen evolving enzyme Photosystem II in chloroplasts: a review. Envir. exp. Bot. 31, 11 21. 2. BEAUREGARD M., GORAJ K., GOFFIN V., HEREMANS K., GOORMATIGHE., RUSSCAERTj.-Ik'l, and MARTIALJ. A. ( 1991 ) Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the cqfl-barrel packing). Protein Engng 4, 745 749. 3. BEAUREGARDM., MORINL. and Poeovlc R. ( 19881 Removal of the 33, 23 and 18 kDa extrinsic proteins ofphotosystem I1 by sulfite treatment at alka-

14.

15.

16.

t-21

line pH. Bioehem. bi@hys. Res. Commun. 152, 612 616. BEAUREGARDM. and PoPovIc R. (19881 Removal of 23 and 18 kDaIton extrinsic polypeptides by sulfate ill photosystem lI particles. J. Pl. Plg, rioL 133, 615 619. BIOU V., (;IBRAT,J. 1"., LEVIN,J. M. and (}ARNIER ,}. 1988) Secondary strueturc prediction: con> bination of three differcnt methods. Protein Fnwg 2, 185 191. BORTItAKURD. and HASEI,KORNR. ( 19891 Nucleotide sequence of the gene encoding the 33 kl)a water oxidizing polypeptide in Anabaena sp. strain PCC 7120 and its expression in E.~cheriehia coll. PI. mo/ec. Biol. 13, 427 439. Bt!I~:NAPR. I, and SHERMAXI, A. {19911 l)clction mutagenesis in @,necho(rstis sp. PCC6803 indicates that the Mn-stabilizing protein of Photosystem II is not essential lbr O., evolution. Biochemi.~trr 30, 44O 446. Cm~y Y. H. and YANt~ .]. T. 1971i A m'w approach to the calculation of secondary structures of glohular proteins by optical rotatory dispersion and circular dichroism. Biochem. biopt{>. Res. Commun. 44, 1285 1291. CnoTma C. i19761 The nature of lhe accessibh' and buried surthccs in proteins..], molec. Biol. 105, 1 [4. CHOU P, Y. and I"ASMANG. D. (19781 Prediction of the se(ondary structure of proteins from their amino acid sequence. ,]. Adz'. Engl,m. 47, 45 148. COHF~NF. E., ABARBANEL R. M., KuxTZ I. 1). and I:LETTERIC, K R. J. (19861 T u r n prediction in proteins using a pattern-matching approach. Biochemimy 25, 266 275. COLEMANW.J. (19901 Chloride binding proteins: mechanistic implications for the oxygen-evolving complex ofPhptosystem II. Photosl,n. Re.~. 23, 1 27. COLEMAX\V..1. and GOVINDJEE (19771 A model tiw the mechanism of chloride activation of oxygen evolution in photosystem I I. Photosyn. Re.~. 13, 199 223. ('RAWFORD I. P., N1ERMANNT. and KmscnN~a K. (19871 Prediction of secondary structure of evolutionary comparison: application to the alpha suhunit of tryptophan synthase. Proteins 2, 118 129. CVTSVOR'rnG. A., ~rHITAKER R. N., HERMANS 1, and I,ENTZB. R. (1989) A new model to describe extrinsic protein binding to phospholipid membranes of varying composition: application to human coagulation proteins. Biochemislrr 28, 7453 7461. EATON-RYEJ.J. and MURATAN. (1989) Evidence that the amino-terminus of the 33 kDa extrinsic

422

M. BEAUREGARD

protein is required fbr binding to the Photosystem II complex. Biochim. bi@@s. Acta 977, 219 226. 17. EMINI E. A., H~rcrIES J. V., PERLOW D. S. and BOGEr~ J. (1985) Induction of hepatitis A virusneutralizing antibody by a virus-specific synthetic peptidc. ,J. l'irol. 55, 836 839. 18. FASMAN G. l). (1991) The prediction of the secondary structure of proteins. Pages 321-332 in H. G. J6RNALL, ed. Metho& in protein ~equenceana/y.~i.~.Birkhauscr, Basel. 19. GARNIERJ., OSC;UTHORPE1). J. and ROBSON B. (1978) Analysis of the accuracy and implications ot" simple methods tbr predicting the secondary structure of globular proteins. ,/. molec. Biol. 120, 97 120. 20. (]HANOrFAKISD. F., BABCOCKG. T. and YOt;UM C. F. (1984) Calcium reconstitutes high ratcs of oxygcn evolution in polypeptide dcpleted Photosystem II preparations. FEB,~"Lell. 167, 127 130. 21. G1BRATJ.-F., (-~ARNIERJ. and ROBSONB. ('.1987) Further developments of protein secondary structure prediction using infbrmation theory. New parameters and consideration of residue pairs. J. mole(:. Biol. 198, 425 443. 22. HAAG E., 1RRGANG K.-D., BOEKEMA E. J. and RENGER G. (1990) Functional and structural analysis of photosystem II core complex ti'oln spinach with high oxygen evolution capacity. Eur. J. Biochem. 189, 47 53. 23. HIe,GINS D. G. and SHARP P. M. (1988) CI,USTAL: a package for pertbrming multiple sequence alignments on a microcomputer. Gene 73, 237 244. 24. HOMANNP. H. (1985) The association of functional anions with the oxygen-evolving center of chloroplasts. Biochim. biophys. Acta 809, 311 319. 25. HOMANNP. H. (1988) The chloride and calcium requirement of photosynthetic water oxidation: effects of pH. Biochim. bi@@s. Acta 934, 1 13. 26. ITOH S. and UWANOS. (1986) Characteristics of the C1 action site in the 02 evolving reaction in PS 1l particles: electrostatic interaction with ions. Pl. (,'ell Phy,~iol. 27, 25- 36. 27. JANINJ., WODAKS., LEVITT M. and MAIC,RET B. (1978) Contbrmation of amino acid side-chains in proteins. ,]. moiec. Biol. 125, 357 386. 28. JANSEN T., ROTHER C., STEPPUHNJ., REINKE H., BEYREUTHER K., JANSSON C., ANDERSSONB. and HERRMANN R. G. (1987) Nucleotide sequence of eDNA clones encoding the complete '23 kDa' and '16 kDa' precursor proteins associated with the photosynthetic oxygen-evolving complex from spinach. FEBS Letl. 216, 234 240. 29. KABSCHW. and SANDER C. (1983) Dictionary of protein secondary structure: pattern recognition of

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

hydrogen-bonded and geometrical ffatures. Biopolymers 22, 2577 2637. KARPLUS P. A. and SCI~ULZ G. E. {1985) Prediction of chain flexibility in proteins. A tool for the selection of peptidc antigens. ;Va&rwi,~sen. 72, 212 213. KAVELAKIK. and GHANOTAK1SD. F. ( 1991 ) Effect of the manganese complex on the binding ot" dm cxlrinsic proteins (17, 23 and 33 kDa) of Photosystem II. Photosyn. Res. 29, 149 155. KOmE H. and |NOUE Y. (1985) Properties of a peripheral 34 kDa protein in @nechococcus vuleanu~ Photosystem I I particles. Its exchangeability with spinach 33 kDa protein in reconstitution of 02 evolution. Biochim. hi@by6. Acla 807, 6+ 73. KYTE J. and DOOLn"I'LE R. 1". (1982) A simple method tbr displaying the hydropathic character of a protein. ,]. molec. Biol. 157, 105--132. LEVINJ. M. and GARNIERJ. (1988) Improvements in a secondary structure prediction method based on a search tbr local sequence homologies and its use as a model building tool. Biochim. biophys. Acla 955, 283 295. MAYFIELD S. P., SCHIRMER-RAHIRE M., FRANK C-., ZURER H. and RocHMx J.-D. (1989) Analysis of the genes ot" the OEE1 and OEE3 proteins of the photosystcm 1I complex from (,Tdamydomonas reinhardlii. PI. molec. Biol. 12, 683- 693. MFADOWSJ. ~'., HULFORD A., RAINES (3. A. and ROmNSON C. (1991 ) Nucleotide sequence of a eDNA clone eneoding the precursor of the 33 kDa protein of the oxygen-evolving complex from wheat. Pl. molec. Biol. 16, 1085 1087. MIYAO M. and MURATAN. (1989) The mode of binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa in the Photosystem II complex of spinach. Biochim. biophys. Acta 977, 315 321. MiYAOM., MURATAN., MAISON-PETERIB., BOE'SSAC A., ETIENNE A.-L. and LAVORELJ. (1987) Effect of the 33-kDa protein on the S-state transition in the oxygen-evolving complex. Pages 1.5.613 616 in J. BmcINS, cd. Progre66 in photosynthesi.~ research. Martinus Nijhoff, Dordrecht. MURATAN. and MIVAOM. (1987) Oxygen-evolving complex of photosystem II in higher plants. Pages 1.5.453 462 in J. Brae,iNS, ed. Progres~ in photosynthesis research. Martinus Nijhott; Dordrecht. NrSHIKAWAK. (1983) Assessment of secondarystructure prediction of proteins. Comparison of computerized Chou-Fasman method with others. Biochim. biophys. Acta 748, 285 299. OH-OKA H., TANAKAS., WADA K., KUWABARA T. and MURATAN. (1986) Complete amino acid sequence of 33 kDa protein isolated from spinach photosystem II particles. FEBS Lett. 197, 63 66.

M O D E L L I N G OF T H E PSII 33 kDa P R ( ) T E I N 42. Oxo T. and INOUE Y. (1984) Ca e+-dependent restoration of O~-cvolving activity in CaCI,,washed PS II particles depleted of33, 24 and 15 kl)a proteins. FEBS Lelt. 168, 281 286. 43. PH1LI~RICK ,J. B. and ZILINSKAS B. A. (1988) Cloning, nuck~otide sequence and mutational analysis c~t"the gene encoding the photosystem I1 mangallese-stabilizing polypeptide of @necho(>ti,~ 6803. Molec. gen. Genel. 212, 418 425. 44. SHDIJ~R A. and MicHVa, H. :1990i Expl'essi(m in E.scherichia coil of the psbO gene encoding the 33 kd protein of the oxygen-evolving complex ti'om spinach. E:IIBO ,J. 9, 1743 1748. t5. SuEx,J.-R. and Ixovl,: Y. (1991 I,oxx pH-induced dissociation of three extrinsic ~roteins t)om O:evolving Photos\stem 11. Pl. (;'ell Ph>iol. 32, 453 457. 46. SHEN ,].-R., SATOtI K. and KATOH S. (1988) (?ah'ium content of oxygen-evolving Photosystem I I preparations from higher plants. Eft'cots of NaC1 treatment. Biockim, hi@to,s. Acta 933, 358 364. 47. STEWART A. (1., I~JUNBER(; U., AKERLUND tf.-E. and ANDF,RSSON B. (1985) Studies on the polypeptide composition of the cyanohacterial oxygenevolving complex. Biochim. biopt!>. Aaa 808, 353 362. 48. STRYNAI)KAN. (i. ,]. and JAMES M. N. (;. 11989) (:rvstal su'uctures of the helix loop helix cahiumbinding proteins. A. Rec. Biochem. 58, 951 99g.

423

49. "I'ANAKA S. arid WADA K. (1988) The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem 1I complexes. Pholosyn. Rc,~. 17, 255 266. 50. TANG X.-S. and SArolt K. (1986) Reconstituti(m of photosynthetic water-splitting activity bv the addition of 33 kDa polypeptide to urea-treated PS I1 reaction center complex. 1,7'2BS Lell. 201, 221 224. 51. VANSPANJIL NI., DIRSKSE ~V. (~'., NAP ,1.-t'. and STn~;KEMA\\:. J. (1991) Isolation and analysis of cI)NA encoding the 33 kDa precursor protein ~;t the oxygen-evolving complex of potato. PI. ,nolec. Bio/. 17, 157 160. 52. VYAS N. K., \:VAS M. N. and Qt;lOCHO F . . \ . (19871 A novel calcium-binding site in the galactose-binding protein of bacterial transport and chemotaxis. Nature 327, 635 638. 53. WAIF,S R., NEWMA,',"B. J., PAPPIN D. and GRAY ,]. C. 19891The extrinsic 3 3 k D a polypeptideof the oxygen-evolving complex of photosystem 11 is a putative calcium-binding protein and ix encoded bv a muhi-gene tamilv in pea. PI, molec. Biol. 12, 439 451. 54. \VEBBER A. N. and GRAY,J. C. (1989) Detection ofcal(ium binding by photosystem I I t)olypeptides immobilized onto nitrocellulose membrane. I"EBS Let/. 249, 79 82.