B:ochimwa et Biophyswa Acta. 1077 (1991) 220-224 . ) 1991 Elsevier Science Publishers B.V. 0167-4838/91/$03.$0 ADONIS 016748389100150M

220

BBAPRO 33879

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum P. H o j r u p i, p. G e r o l a 2.3, H . F . H a n s e n 4, J . M , M i k k e l s e n 5, A . E . S h a h e d L , , J. K n u d s e n 2 p. R o e p s t o r f f ~ a n d J . M . O l s o n 2 t Department of Molecular Biolo~,, Odense University, Odeme (Denmark). z Departments of Biochemistry, Odense University. Odense (Denmark), 3 Department of Ecolo~,. University of Calabria, Arcavacata di Rende. Cosenza (liar). ¢ Department of Clinical Chemistry, Rifshospitalet. Copenhagen (Denmark) and J N ardisk Gcntofie. Oentofie (Denmark) (Received 22 November 1990)

"~ey words: Amino acid sequence; Green photosynthetic bacteria; Light Harvesting Complex

A 7.5-kDa protein has been isolated i'x ~ :~do.osomes of Cklorobium limicekl f. thiosulfatophilum and the complete iwimary structure determined by a t-,,mbinati,m of automatic Edman degradation and plasma desorptton mass spectrometry. The 74-res'due protein shows great homology to a similar protein of unknown function which has been isolated from Pelodietyon htleolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the ehlorosome is discussed.

Intrnduet~ Green photosynthetic bacteria contain the largest light-harvesting systems known among phototrophic organisms [1]. In the green sulfur bacteria, for example, there can be 1000-2000 molecules of light-harvesting bacteriochlorophyll (BChl) c for each reaction center. Almost all the BChl c is found in chlorosomes appressed to the inner surface of the cytoplasmic membrane, whereas the reaction centers are located within the membrane (presumably under the appressed chloroseines). The pathway for en~,'gy transfer is from BChl c to BChl a in the chlorosome, then to BCbl a in the BChl a-protein monolayer attached to the membrane and finally to other BChl a antenna systems in the membr~le ,rod/or to the reaction centers [1-3]. The chlorosome contains in addition to BChl c proteins lipids, and a small amount of carotenoid. Most of the protein is accolmted for by two low molecular weight plaI~peptides: 5.7 kDa in Chloroflexus aurantiacus 141 or 6.3 kDa in green sulfur bacteria [51 and 7.5

* Present ~ddeess: National Organisation for Drug Control aJld Re~¢arch, Cairo. Egypt. Correspondence: p. Hojrup, Department of Molecular Biology, Odense Uni,~rsity. Campusvej 55, 5230 Odense M., Denmark.

kDa in Chiorobium Iimicola f. thiosuifatophilum [6]. It has been assumed that these proteins are involved in the binding of BChl c. but none of these proteins has ever been isolated with any bound BChl c. in fact it has recently been shown that most of the protein can be removed from Chloroflexus chlorosomes without changing the characteristic absorption spectrum of the native BChl c aggregates in the chlorosomes [3,7]. In this paper we refer to these proteins by their molecular masses, so as to avoid unwarranted presumptions about function. The 5.% and 6.3-kDa proteins are about 30% identical, and the 5.7-kDa protein has been localized in the chlorosome envelope by immunogoid labelling (W, Wullink, unpublished data). The 7.5-kDa protein has so far been found only in chlorosomes of green sulfur bacteria and is also an envelope protein (W. Wuilink, unpublished data). In this paper we describe the procedures by which the primary s.ructure of the 7.5-kDa polypcptide from C. limwola f. thiosulfatophilum has been determined, and we discuss the possible rol, of this protein in binding the chiorosome, to the underlying BChl a monolayer. Materials and Method~ The 7.5-kDa protein was purified from chlorosomes of C. [imicola f. thiosulfatophilum. The chlorosomes

221 foil, washed with 0.1% trifluoroacetic acid and subjected to mass spectrometric analysis using a Biolon 10K 2S2Cf plasma desorption mass spectrometer (Biolon, Uppsala, Sweden) [8]. The precision of the mass determination is +0.17o. For all proteins and peptides isolated molecular weights were similarly obtained. Hydrolysis of proteins and peptides were carried out using 6 M HCI containing 0.1¢$ phenol for 18-24 h at l l O ° C in vacuo. Amino acid analysis was carried out on a Beckman 121 MB amino acid analyser [91 for the intact protein, and for the peptides by the PITC method of Heinrikson and Meredith [10]. Automated sequence determination of the intact protein was carri,'d out on a Beckman 890C sequencer while sequence determination of the peptides was carried out on an Applied Biosystems gas phase sequenator using procedures previously described [11 ].

10 3 X M r 43 ~

0

29 - t

o

17.5-- I 12.5-'

6.5-i

Lane

I

2

3

4

5

Fig. 1. SDS-PAGEgel showing purification of chlorosome proteins from Chlorobium Iimicola f. thiosulfatophilum. Lanes contains the followingsamples: l, acetone/water phase after diethyl ether wash: 2, protein standard; 3. total 80~ acetone extract: 4, blank: 5, whole chlorosomes.

were extracted with 80~ acetone at a concentration corresponding to ,4 = 15 cm - t at the BChi c Qy-peak and centrifuged for 4 min at 17 000 × g. The supernatant was mixed with diethyl ether and w.'tter (volume ratio 80~$ acetone/diethyl ether/water, 3 : 5 : 2 ) . The mixture was centrifuged for 10 min at 3 000 × g and the ether phase was removed from the top. The a c e t o n e / water phase was repeatedly washed with fresh ether until the ether wash was colorless. The acetone/water phase was then dried. The entire extraction procedure was carried out in the dark. SDS-PAGE of the acetone extracted chlorosome proteins is shown in Fig. 1. The residue was dissolved in 0.1~ trifluoroacetic acid in isopropanol/water (1 : 1) and loaded onto a Vydac C 4 column (8 × 120 mm) equilibrated with 10~ isopropanol in water containing 0.1~ trifluoroacetic acid and eluted with a 30 min gradient in isopropanol as shown in Fig. 2. The isolated protein was cleaved either with trypsin (10 nmo! s a m p l e + 2 /tg trypsin in 200 pl 0.1 M N H 4 H C O 3, I mM CaCI 2 (pH 8.3), 37°C, 2 h) or with chymotrypsin (15 nmoi sample + 2 pg chymotrypsin in 200/tl 0.1 M N H 4 H C O 3 (pH 8.3), 37°C, 2.5 it). Both digestions were terminated by injection on HPLC. The peptides were separated on a reverse-phase column packed with Nucleosil ODS (4 x 250 mm, 1 0 / t m particle sge) using a 20 rain linear gradient from 5 to 45~ of isopropanol in 0.1~ trifluoroacetic acid. During the enzymatic digestions small aliquots (50100 pmol) were withdrawn from the reaction volumes and appfied to nitrocellulose covered aluminized mylar

Results

Purification of the 8Chl-free 80~ acetone extract by reverse-phase HPLC resulted in the elution of a minor peak (peak 1) and a major double peak (peaks 2a and 2b, Fig 2). SDS-PAGE gel electrophoresis of both the minor and the major peaks showed bands in the 4 - 5 kDa region, which could not be resolved. Amino acid analysis of the 7.5-kDa proteins from the two major peaks showed them to be almost identical, while the protein associated with the minor band differed considerably (Table I). The protein eluting as the minor peak (peak 1) seems identical to the 6.3-kDa polypeptide reported by Wagner-Huber et al. [5] as the moleca!ar weight and amino acid composition as calculated from the partial sequence in [5] are compatible with Table I (peak 1). A

211

I%a 100

.f 5o

10

l'mw0r~

20

30

o

Fig. 2. HPLC :;eparadon of proteins isolated by a~tone extraction of chlorosomes. The 6.3-kDa protein is found in peak I and the 7.5-kDa protein is found in peaks 2a and 2b. Separation was carried out on a Vydac C4 column with a gradient of isopropanol in 0.l~ trifluoroacetic acid as indicated by the dotted line.

222 short sequence run on the intact protein showed the N-terminal 22 residues to be identical to the 6.3-kDa protein. Mass spectrometric analysis with PDMS of the two components of the double peaks 2a and 2b. showed them to have average molecular masses of 7488.2 ± 7.5 Da and 7483.0 ± 7.5 Da respectively. The protein in peak 2b was shown by mass spectrotnetric peptide mapping (Fig. 3a, 3b) to deviate from that in peak 2a in a single peptide only. later shown to be the N-terminal peptide. This peptide was determined to have a molecular mass approx. 18 Da less than the corresponding peptide from peak 2a. As sequence determination of the intact protein 2b as well as of the N-terminal tryptic peptid¢ of protein 2b was unsuccessful, it was concluded that protein 2b contains a modified N-terminal

residue. The exact ,ature of this modification has not been determined. The apparent blocking of protein 2b might be explained by a cyclisation of Asn-2-Gly-3 to form an imide. The resulting loss of NH~ is within the range of molecular weight difference. However, upon sequencing this should show the N-terminal serine residue which was not the case. Based on the peptide maps in Fig. 3 and the near identity of the amino acid compositions, protein 2a and 2b were concluded to be identical except for the N-terminal modification. The reason for the mass analysis of the intact molecule 21) not showing a mass 18 Da less than that of 2a could be that part the preparation contained an oxidized methionine (see below). Automated Edman degradation of the intact 7.5-kDa protein (2a~ allowed the unambiguious determination of

1412

1609.0

105~

7O(

(3

N

1125

1750

2375

3000

M/Z 805 t~47

1~9.5

603

~40~ 0

(3 201

ll 112s

I

I

IL I~S)

'

~

,~

a A ~'Ts

"

~mo

M/Z Fig. 3. Plasma desorption mass spectrum of tryptic peptide maps of the 7.5-kDa protein. (a) Protein isolated from peak 2a, Fig 2. (b) Protein

isolated frompeak 2b, Fig. 2.

223 TABLE I

Amino m~id ~Tmposttion and mo!ecular .'eight of the major cld~'~Js,~me proteins .from Chlorol~um hmw~do f, thwsulfat~q~hdum Experimental values are means of three HCI hydroly~s. -., below detection limit. Amino acid

Asx Thr Set" GIx Pro Gly Ala

Cys Val Met

Composititm (mol of ~sidue/mol) acid hydrolysis o4"1

acid hydrolys,s of 2a

acid hydrolysis of 2b

sequence analysis of 2a

6.9 5.4 8.3 6.4

8.7 7.3 2.9 6.7 2.2 6.0 14.2

8.6 8.2 3.2 6.8 2.4 6.1 14.5

9 8 3 6 2 6 14

24.2 7.0 -

-

-

9.0 8.3

5.3 0.5

5.0 0.8

lie Leu Tyr Phe Lys His Arg Trp

4.7 6.6 1.5 3.6 2.6 1,6 3.9 n.d.

4.1 8,4

3.3 7.5

2.9 4.8

2.7 4.1

n.d.

n.d.

M,

6220.5

748&2

7483.0

0 5 ! 4 8 0 3 5 0 0 0 7488.5

the first 37 residues (Pig. 4). Digestion of the protein with trypsin for I h and peptide mapping of the reactie-~ mixture using PDMS shows the presence of four peptides (Fig. 3a). Prolonged digestion time shifted the peak at m / z 2745 + 12.7 to m / z 2616 + 12.6 corresponding to the loss of a C-terminal lysine residue (this can be seen in Fig 2b). The peptide giving rise to the MH + peak at m / z 1609+ 11.6 had an expected m / z of 1592.1 + 11.6 as calculated from the sequence data. The differettce in mass (10.9 Da) was shown to be caused by oxidation of the methionine residue as the expected mass could be obtained by reduction of the peptide with 0.775 M dithiothreitol for 18 h at 37°C.

By adding up the masses of the four peptides and subtracting the mass of three water" molecules introduced by the enzymatic cleavages and the mass of an oxygen atom from the oxidized methionine, a value for the mass of the intact molecule of 7491.9 + 7.5 Da is obtained. This value is within the experimental error of the mass obtained of the intact molecule (7488,3 Da), and shows that the four peptides in total constitute the complete sequence. All of the tryptic peptides were sequenced, but the C-terminal peptide could only be sequenced up to residue 64. Another sample of the protein was then digested with chymotrypsin and the C-terminal chymotryptic peptide was identified by comparing PDMS analysis of the HPLC-purified fractions with the sequence already determined. All 11 residues of this peptide were sequenced. Although only a one residue overlap was obtained between the tryptic peptide T2 and the chymotryptic peptide CI, mass spectrometric data of the peptides and the intact protein shows the sequence in Fig. 4 to be correct. The mass calculated based on the determined sequence (7488.5 Da) i,~ in agreement with that measured for the intact protein. Discussion The primary ~tructure of the 7.5-kDa protein from the chlorosomes of C limicola shows homology neither to the 6.3-kDa protein nor to the 5.7-kDa protein isolated from chlorosomes of Chloroflexus aurantiacus [~,,6] (best match shows only 17.6% identity). However a similar protein has been isolated from Pelodictyon luteolum by Wagner-Huber et al. [5] who sequenced the 24 N-terminal residues (of which 21 were identical to the 7.5-kDa protein of C. limicola). The protein could be isolated from whole cell extracts but not from isolated chlorosomes. No function has been ascribed to this protein. A search of the Protein Identification Resource database (version 26) did not reveal any other significant homologies. An interesting phenomenon is the occurrence of four internal homologous regions of ten/eleven residues.

Ser-A~n-Gly-Thf-Asn-IIe-Asp-Val-Ala-Gky-AIa-Zle-Asn-Thr-Leu-Thr-GIu-Thr-Phe-GI¥

20

L•s-Leu-•he-G•n-•et-G•n-Leu-A•p-Va•-A•a-Asn-Thr-A•a-Leu-Lys-A•a-Le•-A•a-Asp-Va•-

40

TI'~

-qP ~'~ " ~

-,o

A~a-G1u-~r~-Leu-G~¥-L~s-Thr-A~a-Thr-Asp-L~u-~a~-~1y-Asn-~he-A1a-G~-A~a-A~a-Thr-

60

Gln-lle-Leu-Gln-Ser-Va1-Ser-Ala-A1a-Ile-Ala-Pro-Lys-Lys

74

¢1-* --* --* --'P -'*'"~ " ~ " * "*" " * " ~ Fig. 4. Sequenceof the 7.5-kDa protein from the chlorosomeof Chlorobiumlimwola f. thiosulfatophilum.Only peptides used in the assemblyof the sequenceis shown. Residues identifiedby ~oem,ing are underlined by arrows. TI and T2 are trypti¢ t,-,,~tides,CI is a chymotrypticpeptide.

224 SNGTNIDVAG-AINTLTETFGKLFOM~L DVANTALKALA DVAEPLGKTAT ~LVGNFAGAArOIL~SVSAAIAPKK

Fig. 5. Imernal homology of the 7.5-kDa protein. Identical residues are ~xed.

With a single insertion in the first of these regions they can be arranged with an identity between 18 and 45~ (Fig. 5) and seem to represent gene duplications. The distance between the first and the second homology region is eleven residues and could represent a fifth duplication, but the similarity to the other regions is too small to show any relationship. Each homologous region begins with a negatively charged Asp residue on the N-terminal side. The exhaustive analysis of the secondary structure by Gerola e t a l . by the method of Chou and Fasman [6] predicted the protein to be made up of 5 a-helices (corresponding to 69~ of the protein) and a ~ b e n d as shown in Fig. 6. The presence of a high content of a-helix was supported by drawing helical wheels of the proposed a-helical regions [61. All five regions were clearly amphipatic with the hydrophobic residues localized to one side and the hydrophilic residues localized to the other. One exception is Gin26 which is situated in the hydrophobic arc of the second a-helix. Comparison of the predicted structure with the homologous regions shows the fast region (Asp-7-Thr-16) to contain the end of the first a-helix (Asn-5-11e-12) while the next three homologous regions are similar in containing the end of an a-helix and the beginning of the next a-hefix. The proposed gene duplications thus seems to favour the more exposed parts of the molecule suggesting that the duplicated regions could contain functions important for interactions with the surroundings. Gerola et al. [6] original!y proposed that the 7.5-kDa protein was the Bchl c-binding protein of the chlorosome, but there is no evidence that either the 6.3- or 7.5-kDa protein binds BChl c [3,7,13]. Since the 7.5-kDa

5 10 15 20 25 30 35 I-0 SNGTNI DVAGA I NT LT ET FGKLFOMOLDVANTALKALADVA--E ~

~

p I

,~

,Oooqqooooooooo_~

LI

KKPA I AASVSOL I OTAAGAFNGVLDTATK - - O /*5 '7O 65 6O 55 5O

Fig. 6. Secondary structure predicted for the 7.5-kDa protein [4]. the symboilf'tnfindicates a-helix and the symbol---~.- indicates/g-sheet. There is one B-turn (GIn-42 to Gly.45). Adapted from Gerola et aL [6].

protein is localized in the chlorosome envelope as shown by the use of gold-labelled antibodies (W. Wullink, unpublished data), we suggest that this protein may be part of the baseplate [14] which binds the chlorosome to the attachment site on the cytoplasmic membrane. In the green sulfur bacteria this attachment site consists of a two-dimensional crystal of Bchi a-protein [14], the surface of which contains charged amino acid residues (Glu-, Asp-, Lys + and Arg+). We suggest that the 7.5-kDa protein may bind the chlorosome to the BChl a-pro[c[, monolayer by salt bridges between A s p - residues in the 7.5-kDa protein and Lys + a n d / o r Arg + residues in the BChl a-protein [15,16]. We further suggest that the four homologous regions of the 7.5-kDa protein contain the binding sites of the chlorosome (the Asp residues are found only in these four regions).

References 10lson, J.M. (1980) Biochim. Biophys. Acta. 594, 33-51. 2 van Dorsen, RJ.. Gerola, P.D, Olson, J.M. and Amesz J. (1986) Biochim. Biophys. Aeta 848, 77-82. 3 Griebenow, K. and Holzwarth, A.R. (1990) in Molecular Biology of Membrane-Bound Complexes in Photosynthetic Bacteria (Drews. G. and Dawes, E.A., eds.), pp. 375-381. Plenum Press, New York. 4 Wechsler, T., Surer, F., Fuller, R.C. and Zuber, H. (1985) FEBS Let[. 181, 173-178. 5 Wag#~er-Huber, R., Brunisholz, R., Frank, G. and Zuber, H. (1988) FEBS Let[. 239, 8-12. 6 Gerola, P.D., Hejrup, P., Knudsen, J., Roepstorff. P. and Olson, J.M. (1988) in Green Photosynthetic Bacteria (Olson, J.M, Ormerod, J.G.. Am..~. J., Stackebrandt, E. and Triiper. H.G., eds.), pp. 43-52. Plenum. New York. 7 Holzwarth, A.R.. Griehenow, K. and Schaffener, K. (1990) Z. Natarforsch. 45c, 203-206. 8 Nielsen, P.F., Klarskov, K., Hejrup, P. and Roepstorff, P. (1988) Biomed. Environm. Mass Spectrom. 17, 355-362. 9 Petersen, T.E.. Thogersen, H.C., Sourup-Jensen. L., Magnusson, S. and JOrnvall, H. (1980) FEBS Lett. 114, 278-282. 10 Heinrikson, R.L. and Meredith, S.C. (1984) Anal, Biochem. 136, 65-74. 11 Hejrup, P., Andersen, S.O. and ReepstorfL P. (1986) Biochem. J. 236, 713-720. 12 prevelige, P. Jr. and Fasman, G.D. (1989) in Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G.D., ed.), pp. 391-416, Plenum Press, New York, 13 Olson. J.M.. Brune, D.C. and Gerola, P.D. (1990) in Molecular Biology of Membrane-Bound Complexes in Photosynthetic Bacteria (Drews. G. and Dawes, E.A., eds.), pp. 227-234, Plenum press, New York. 14 Olson, J.M. (1988) in Green Photosynthetic Bacteria (Olson, J.M., Ormerock J.G.. Amesz, J.. Stackebrandt, E. and Trfiper, H.G., eds.), pp. 1-2, Plenum, New York. 15 Mat[hews, B.W., Fenna, R.E., Bolognesi, M.C., Schnfid, M.F. and Olson, J.M. (1979) J. MOl. Biol. 131,259-285. 16 Daurat-Larroque. S.T., Brew. K. and Fenna. R.F,, (1986) J. Biol. Chem. 261.3607-3615.