- Email: [email protected]
Reconstitution and immobilization of photo-reaction units from photosynthetic bacterium Rhodopseudomonas viridis
Materials Science and Engineering C 6 Ž1998. 285–290
Reconstitution and immobilization of photo-reaction units from photosynthetic bacterium Rhodopseudomonas Õiridis Shu-ichi Ajiki b
a,)
, Hiroaki Sugino a , Hideki Toyotama a , Masayuki Hara
b,c
, Jun Miyake
b,c
a Tsukuba Research Laboratory, Stanley Electric Co. Ltd., 5-9-5 Tokodai, Tsukuba, Ibaraki 300-2635, Japan National Institute for AdÕanced Interdisciplinary Research, AIST, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan c National Institute of Bioscience and Human-Technology, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Received 10 May 1998
Abstract Photo-reaction units ŽPRUs. were solubilized from chromatophores of the photosynthetic bacterium Rhodopseudomonas Õiridis under high salt concentration using Triton X-100 as a detergent. The absorption spectrum of isolated PRUs was similar to that of native chromatophores. Isolated PRUs were composed of reaction centers ŽRCs. and light-harvesting ŽLH. proteins. Purified PRUs were reconstituted into liposomes. The Q Y absorption bands derived from bacteriochlorophyll ŽBChl. b in LH subunits of PRUs were red-shifted from 1000 nm in the isolated PRUs solution to 1006 nm in the reconstituted liposomes, contrasting with the 1015 nm for native PRUs in chromatophores. To make a photo-electrochemical conversion layer for photocell or biosensor, we deposited PRU liposomes on a solid substrate by electrodeposition. The orientation angle between the molecular axis of PRUs and the disk normal was 2.48 for the deposited PRU liposomes, compared with 20.58 for purified chromatophores deposited. This demonstrates that the vertical alignment of protein molecules with the disk normal was superior for electrodeposited protein molecules immobilized using PRU liposomes compared with those using purified chromatophores. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Rhodopseudomonas Õiridis; Photo-reaction unit; Immobilization; Orientation angle
1. Introduction Purple nonsulfur photosynthetic bacteria contain two types of LH protein complexes. The first type, LH1, surrounds the RC and these make a large protein complex, called a PRU. The second type, LH2, is associated with the PRU and is present in variable amounts in chromatophore membranes. The photosynthetic bacterium Rps. Õiridis we used has only LH1-type protein complexes w1x. The RCs of purple bacteria are widely used to study the key mechanism of photosynthesis: charge separation in BChl b dimer and subsequent electron transfer to primary quinone. LH protein complex binds around RC to transfer the light energy from antenna pigments in LH protein complex to BChl b dimer in RC. We previously made a photocell w2,3x and herbicide
)
Corresponding author. Tel.: q81-298-478883; Fax: q81-298474165; E-mail: [email protected]
sensor w4,5x from a combination of chromatophores, PRUs, and RCs. LH proteins have many chromophores, and the orientation of the Q Y moments of the chromophores are closer to the perpendicular than Q X moments w6x. The cross-section of the absorption moment is maximized for the incident light parallel to the molecular axis. In the chromatophore membranes, RCs transfer electrons across a lipid membrane vertically and unidirectionally. To facilitate this transfer, it is therefore critical to immobilize and manipulate photosynthetic proteins at a desired orientation on a substrate. In this study, first we reconstituted PRUs into liposomes and immobilized PRU liposomes on the solid substrate. The electrodeposited protein molecules immobilized using PRU liposomes were more vertical to the substrate than those immobilized using purified chromatophores. The absorption moments were parallel to the substrate and could therefore absorb effectively with directivity. We then demonstrated PRU liposomes as adequate materials for immobilization by electrodeposition.
0928-4931r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 9 8 . 0 0 0 6 4 - 2
286
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
2. Materials and methods 2.1. Preparation of PRU Chromatophores from Rps. Õiridis were prepared as described by Ajiki et al. w2x. The A 1015 Žabsorbance at 1015 nm. of a chromatophore suspension was measured, and a portion of suspension was mixed with the appropriate amount of Triton X-100 Žfrom a 20% stock solution. and salt to yield a final solution volume of 2 ml that contained A1015 s 150, 5% Žwrv. Triton X-100, and 1 M salt in 10 mM sodium phosphate buffer ŽpH 7.0.. After the solution was incubated for 30 min, it was centrifuged for 15 min at 260,000 = g, and the supernatant was applied to a Sepharose CL-6B ŽPharmacia, Sweden. gel filtration column Ži.d. 2.5 = 100 cm.. The column was equilibrated with a solution of 0.1% Triton X-100, 150 mM LiCl, and 0.02% NaN3 in 10 mM sodium acetate buffer ŽpH 6.0., and then PRUs were eluted with the same solution. All of the above procedures in the PRU preparation were done under dark conditions and at 48C. The PRU fractions were pooled and then concentrated by ultrafiltration ŽDIAFLO PM30, Amicon Div., USA.. The detergent was changed from Triton X-100 to CHAPS-acetate Ž0.1% CHAPS, 150 mM LiCl, and 0.02% NaN3 in 10 mM sodium acetate, pH 6.0. for dialysis. PRU sediments were obtained by centrifuging the PRU solution concentrate for 150 min at 417,000 = g. After the supernatant was removed, the PRU sediments were resuspended in CHAPS-acetate. 2.2. SDS-PAGE SDS-PAGE analysis was done according to Laemmli w7x with slight modification. A 12.5–20% gradient gel was used as separation gel. After the electrophoresis, the gel was stained with Commassie Brilliant Blue G-250. 2.3. Analysis of chromophores Chromophores of the PRUs were analyzed as described by Hara et al. w3x after extraction with acetonermethanol Ž7r2 vrv.. The absorbance was measured at 790 nm, and the BChl b concentration was determined using an extinction coefficient of 122 mMy1 cmy1 reported by Garcia et al. w8x. For the extinction coefficients for the carotenoids, we used the values given by Malhorta et al. w9x: 169 mMy1 cmy1 at 436 nm for neurosporene and its dihydro derivative, and 185 mMy1 cmy1 at 468 nm for lycopene and its dihydro derivative. 2.4. Determination of protein concentration Protein concentration was determined by the method of Lowry et al. w10x. When Triton X-100 was present in the
solution, 0.5% SDS was added to prevent the interference of color development described by Dulley and Grieve w11x. Bovine serum albumin was used as the protein standard. 2.5. Preparation of stock lipid solution For reconstitution into liposomes, we prepared a stock lipid solution as follows. Phosphatidylethanolamine ŽPE; from E. coli, Type IX, Sigma, USA. was used as a phospholipid. Acetone-washed PE Ž40 mg. was dispersed in 4 ml of 50 mM K-HEPES ŽpH 7.8. containing 0.5% deoxycholate, and then sonicated to clarity on ice under a constant stream of nitrogen gas using a probe-type sonicator ŽSONIFIER 450, Branson, USA.. The lipid solution was stored in 1-ml aliquots in a deep freezer Žy808C.. Before use, this stock lipid solution was thawed to room temperature. 2.6. Reconstitution of PRU into liposomes PRUs were reconstituted into liposomes by a dialysis method as described by Molenaar et al. w12x and Crielaard et al. w13,14x with slight modification. PRU solution was added to a PE stock solution to yield a molecular ratio of phospholipids to PRU of 1000:1. After mixing, the solution was dialyzed at 48C for 16–20 h against a 250-fold volume of 10 mM K-HEPES ŽpH 7.8., during which the K-HEPES buffer was changed three times. After dialysis, the liposomes were centrifuged for 10 min at 81,000 = g and resuspended in 5 mM sodium phosphate ŽpH 7.0. by sonication with the probe-type sonicator. The yield of reconstitution was determined by comparing the adsorption spectrum of the supernatant after centrifugation with that of the resuspended solution before centrifugation. 2.7. Electrodeposition of PRU liposomes and chromatophores PRU liposomes and chromatophores were immobilized on a solid substrate electrode by electrodeposition as described by Ajiki et al. w2x. Indium–tin–oxide ŽITO. coated glass ŽGeomatec, Yokohama. was used for the optically transparent substrate electrode Ž2.5 = 25 = 1.1 mm.. Chromatophores were purified for electrodeposition by a sucrose density gradient as described by Jacob and Miller w1x with slight modification. Chromatophores were overlaid onto a discontinuous gradient, which was single layers of 0.5, 1.0, 1.5, and 2.0 M sucrose solutions dissolved in a 10 mM sodium phosphate ŽpH 7.0.. The gradient was centrifuged for 150 min at 417,000 = g, resulting in a purified chromatophore fraction collecting at the interface of the 1.0 and 1.5 M sucrose steps in the
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
287
gradient. The purified chromatophores were washed twice with 5 mM sodium phosphate and then resuspended in the same phosphate buffer adjusted to A1015 s 100. 2.8. Linear dichroism measurement of LH on a solid substrate We determined the orientation by measuring the linear dichroism ŽLD. of chromophores as described by Jacob and Miller w1x, Breton et al. w15x, Nabedryk and Breton w16x, and Breton w17x. The extent of orientation of the PRUs or chromatophores deposited on the ITO electrode was monitored by measuring the LD of the Q Y transitions of BChl b in the LH protein subunits Žat 1006 nm for PRUs or at 1015 nm for chromatophores.. The LD spectra were recorded using a spectrophotometer UV-3101PC ŽShimadzu, Kyoto. that had a polarizer. The light beam used in this measurement was polarized either perpendicular to the normal to the disk plane Žto record A 0 . or parallel to the plane of incidence Žto record A 90 .. Polarized absorption spectra were measured by tilting the sample to 608 with respect to the direction of the beam. Adsorption spectra of the unpolarized light Žto record A N . were recorded without tilting the sample.
3. Results 3.1. Solubilization of chromatophores We studied the effects of salt concentration and type ŽLiCl, NaCl, and KCl. on the solubilization of protein from chromatophores. The yield of solubilization using each salt was estimated from the protein concentration, because the absorption spectrum and SDS-PAGE analysis showed that the solubilized proteins had the same polypeptide composition for all salts. The initial protein concentration was about 12.7 mgrml. KCl was more effective than the other salts in solubilizing PRU ŽFig. 1.. Compared with the protein solubilization in the absence of salt, the addi-
Fig. 2. Absorption spectrum of PRU. Absorption spectrum of ŽŽA. dotted line. native chromatophore, ŽŽB. dashed line. isolated PRU solution, and ŽŽC. solid line. PRU liposomes solution. The peak around 1015 nm in the chromatophore spectrum is slightly shifted to about 1000 nm in the PRU spectrum. The peak around 680 nm in the PRU fraction may be derived from BChl b hydrate.
tion of 1 M KCl increased the solubilized protein concentration by a factor of 2.8. The stability of solubilized PRU was also affected by the salt type and by the pH of the buffer Ždata not shown.. PRU was more stable in buffer that contained LiCl than in those containing the other salts. We therefore used KCl for the solubilization and LiCl was for the elution. 3.2. Preparation of PRU The spectra of the chromatophores and the pooled PRUs ŽFig. 2A and B, respectively. show that the peaks derived from carotenoids and BChl b in PRUs are similar to those from chromatophores. However, the absorbance maximum of BChl b at 1015 nm, derived from BChl b in the LH proteins of chromatophores, was shifted to around 1000 nm in the PRU fraction. BChl b monomer Ž830 nm in RC. and BPhe b are distinguishable in the PRU fraction but not in the chromatophores. The peak around 680 nm in the PRU fraction may be derived from BChl b hydrate. 3.3. Characterization of PRU
Fig. 1. Effect of salt concentration on PRU solubilization. The amount of solubilized protein as a function of salt concentration was determined by the Lowry method. Initial protein concentration was 12.7 mgrml.
The SDS-PAGE analysis of the polypeptide composition of the PRUs ŽFig. 3. shows that the main components of the PRU fraction was RC ŽH, M, L, and Cyt C. and LH proteins Ž a , b, and g ., with trace amounts of other polypeptides. A relative molecular mass Ž Mr . of the PRU Žestimated using HPLC gel filtration. was about 610 kDa in the presence of Triton X-100 Ždata not shown.. For determining the PRU concentration, we used an extinction coefficient of 4.3 mMy1 cmy1 at 1000 nm for BChl b in LH proteins w3x. The calculated number of
288
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290 Table 1 Materials
S
SRC
w Ž8.
Samples
PRU liposomes Chromatophores
y0.327 y0.265
y0.998 y0.816
2.4 20.5
24 21
isolated PRU solution, but recovered to 1006 nm for the PRU liposomes solution. 3.5. LD of immobilized PRUs
Fig. 3. Electrophoresis patterns from SDS-PAGE analysis. Lanes 1, 2, and 3 are chromatophores ŽChr., isolated PRUs, and RCs respectively. Lane 4 is the molecular marker ŽSTD.. The primary components of PRU are RC ŽH, M, L and Cyt C. and LH Ž a , b and g ., with trace amounts of polypeptide.
chromophores per PRU was 27.8 for BChl b and 16.5 for carotenoids. 3.4. Reconstitution of PRUs into liposomes PRUs were reconstituted into PE liposomes using dialysis. The yield of reconstitution was 80% Ždata not shown., which we determined by centrifuging the solution of PRU liposomes. For the chromatophores solution, the peak of the BChl b Q Y moment for LH ŽFig. 2A. occurred at 1015 nm. This peak was shifted to about 1000 nm for the
We measured the LD of immobilized PRUs on ITO substrates to evaluate an alignment of protein molecules. In a quantitative analysis of the orientation distribution of transition moments, Breton reported that for the transition moment M Žcorresponding to one absorption band. oriented with respect to the normal N to the disk, the orientation distribution can be characterized by an order parameter S: Ss
1 y 3 cos 2w
Ž 1.
2
where w is the angle of the orientation distribution between N and the transition moments measured M ŽFig. 4.. The LD of polarized light and the absorbance of unpolarized light is A 0 y A 90
1 s
AN
2'3
=
3S 1qS
.
Ž 2.
Here, S is S s SRC = S LH
Ž 3.
where SRC is the distribution of the two-fold symmetry axis of RC with respect to N and S LH is the distribution of the Q Y transition moments of antennae BChl b in LH proteins. The angle of the Q Y transition moments is 208 with respect to the membrane plane w1x, thereby making it 708 with respect to the two-fold symmetry axis of RC. By Eq. Ž1., therefore the calculated S LH is 0.325. For PRU liposomes electrodeposited on ITO substrates, the measured S was y0.327 Ž N s 24., and the calculated SRC was y1.008. From Eq. Ž1., the range of S is from y1.0 to 0.5. When the angle of this Q Y transition moment was changed from 708 to 70.28 with respect to the two-fold symmetry axis of RC, the calculated SRC is y0.998 and w is 2.48. The following Table 1 lists the results for the measured S and calculated SRC for the electrodeposited PRU liposomes and chromatophores.
4. Discussion Fig. 4. Orientation angle of PRU. Model for the orientation of the transition moment, M. Transition moments of the Q Y band for BChl b in LH make an angle w with the disk normal N.
We previously constructed photoelectric devices and herbicide sensor using chromatophores, PRUs, and RCs.
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
To immobilize photosynthetic proteins on a solid substrate, we tried several methods for controlling the molecular orientation; Langmuir–Blodgett ŽLB. film method, electrodeposition, and the avidin–biotin method w21x. Manipulation and immobilization of photosynthetic proteins at a specific orientation on a substrate is critical because RCs transfer electrons across a lipid membrane vertically and unidirectionally. Our results in this study show that the yield of solubilization is improved by the addition of salt along with Triton X-100. Fig. 1 shows that the amount of total solubilized protein increased with increasing salt concentration. This enhancement depended on the type of salt; KCl ) NaCl ) LiCl. This order of enhancement is the same as the order of the ionic radius of each cation. The addition of NaCl helps to solubilize membrane receptors with ionic detergent w18,19x. However, Triton X-100 is a non-ionic detergent. The reason solubilization is affected by the addition of salt may be the effect of the change in ionic strength or the effect of chaotropic ion-like. Further study is needed to clarify the reason. Using gel filtration, we estimated the Mr of the PRU to be about 610 kDa. Considering that an equal weight of detergent is bound to the membrane protein and that the molecular weight of PRU was estimated by Hara et al. to be about 368 kDa w3x, PRU may be monodispersed in the detergent micelle. Using the extinction coefficients of BChl b and PRU, we estimated the number of BChl b and carotenoid molecules per PRU to be 27.8 and 16.5, respectively. Stark et al. w20x reported that twelve subunits of LH protein, where each subunit has three polypeptides Ž a , b and g ., bind around RC and each subunit contains two BChl b molecules. Therefore, one unit of PRU has 28 BChl b molecules: 4 in the RCs and 24 in the LH proteins. Assuming one carotenoid molecule is contained in the RC and twelve molecules are contained in the LH proteins, the expected number of chromophores per PRU is 28 molecules of BChl b and 13 molecules of carotenoids. The slight peak shift in the isolated PRUs solution around 1000 nm suggests that a slight hydrophobicity or conformational change in the LH proteins occurred by the binding of the detergent rather than the lipid bilayer to the hydrophobic region of PRU or by denaturation of the protein due to the solubilization. This peak shift was not recovered enough by the reconstitution into liposomes. The LH of PRU was distorted by the solubilization, and the peak of BChl b in the LH was shifted to 1006 nm. In the LD analysis, this distortion was taken into account in the calculations. Electrodeposited protein molecules immobilized using PRU liposomes had better vertical alignment compared with those using purified chromatophores. Jacob and Miller w1x reported that the lipids protein ratio of chromatophores is 0.56 Žmg lipidsrmg proteins.. The lipids protein ratio of the PRU liposomes that we prepared was about 1.9 Žmg
289
lipidsrmg proteins.. Compared with chromatophores, PRU liposomes are a relatively ‘soft material’. In this study, we achieved vertical immobilization of PRU by combining a soft material and an electric field.
5. Conclusion Photosynthetic proteins have many chromophores, and the absorption moments Žespecially Q Y moments. are oriented perpendicular to the molecular axis. The crosssection of each absorption moment is maximized for the incident light parallel to the molecular axis. We demonstrated that the vertical alignment of protein molecules with the disk normal was superior for electrodeposited protein molecules immobilized using PRU liposomes compared with those using purified chromatophores. When PRU liposomes were used, the absorption moments were parallel to the substrate and therefore could absorb effectively with directivity. We demonstrated PRU liposomes as adequate materials when immobilization is done by electrodeposition.
Acknowledgements This work was supported in part by the project ‘Research and Development of Protein Molecular Assembly’, which is supported by an R & D Project of Basic Technology for Future Industry, under the Agency of Industrial Science and Technology ŽAIST., Ministry of International Trade and Industry ŽMITI.. We express our thanks to our colleagues at Stanley: Mr. Masami Kumei and Mr. Yoshiaki Yasuda for helpful discussions, and Mr. Yasuhito Ikeda for technical assistance.
References w1x J.S. Jacob, K.R. Miller, Arch. Biochem. Biophys. 223 Ž1983. 282. w2x S. Ajiki, H. Toyotama, M. Hara, J. Miyake, Bioelectrochem. Bioenerg. 43 Ž1997. 71. w3x M. Hara, K. Namba, Y. Hirata, T. Majima, S. Kawamura, Y. Asada, J. Miyake, Plant Cell Physiol. 31 Ž1990. 951. w4x T. Tamura, A. Sato, M. Hara, Y. Asada, J. Miyake, Biotechnol. Tech. 8 Ž1994. 615. w5x H. Peters, C. Schmidt-Dannert, R.D. Schmid, Mater. Sci. Eng. C 4 Ž1997. 227. w6x G. Paillotin, A. Vermeglio, J. Breton, Biochim. Biophys. Acta 545 Ž1979. 249. w7x U.K. Laemmli, Nature 227 Ž1970. 680. w8x A. Garcia, L.P. Vernon, B. Ke, H. Mollenhauer, Biochemistry 7 Ž1968. 326. w9x H.C. Malhorta, G. Britton, T.W. Goodwin, Int. J. Vit. Res. 40 Ž1970. 315. w10x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 Ž1951. 265. w11x J.R. Dulley, P.A. Grieve, Anal. Biochem. 64 Ž1975. 136.
290
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
w12x D. Molenaar, W. Crielaad, K.J. Hellingwerf, Biochemistry 27 Ž1988. 2014. w13x W. Crielaad, K.J. Hellingwerf, W.N. Konings, Biochim. Biophys. Acta 973 Ž1989. 205. w14x W. Crielaad, F. van Mouric, R. van Grondelle, W.N. Konings, K.J. Hellingwerf, Biochim. Biophys. Acta 1100 Ž1992. 9. w15x J. Breton, M. Michel-Villaz, G. Paillotin, Biochim. Biophys. Acta 314 Ž1973. 42.
w16x E. Nabedryk, J. Breton, Biochim. Biophys. Acta 635 Ž1981. 515. w17x J. Breton, Biochim. Biophys. Acta 810 Ž1985. 235. w18x R.D. Howells, T.L. Gioannini, J.M. Hiller, E.J. Simon, J. Pharmacol. Exp. Ther. 222 Ž1982. 629. w19x M. Maruyama, H. Sugino, K. Akita, H. Hatanaka, Brain Res. 401 Ž1987. 14. w20x W. Stark, F. Jay, K. Muehlethaler, Arch. Microbiol. 146 Ž1986. 130. w21x J. Miyake, M. Hara, Mater. Sci. Eng. C 4 Ž1997. 213.
Reconstitution and immobilization of photo-reaction units from photosynthetic bacterium Rhodopseudomonas Õiridis Shu-ichi Ajiki b
a,)
, Hiroaki Sugino a , Hideki Toyotama a , Masayuki Hara
b,c
, Jun Miyake
b,c
a Tsukuba Research Laboratory, Stanley Electric Co. Ltd., 5-9-5 Tokodai, Tsukuba, Ibaraki 300-2635, Japan National Institute for AdÕanced Interdisciplinary Research, AIST, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan c National Institute of Bioscience and Human-Technology, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Received 10 May 1998
Abstract Photo-reaction units ŽPRUs. were solubilized from chromatophores of the photosynthetic bacterium Rhodopseudomonas Õiridis under high salt concentration using Triton X-100 as a detergent. The absorption spectrum of isolated PRUs was similar to that of native chromatophores. Isolated PRUs were composed of reaction centers ŽRCs. and light-harvesting ŽLH. proteins. Purified PRUs were reconstituted into liposomes. The Q Y absorption bands derived from bacteriochlorophyll ŽBChl. b in LH subunits of PRUs were red-shifted from 1000 nm in the isolated PRUs solution to 1006 nm in the reconstituted liposomes, contrasting with the 1015 nm for native PRUs in chromatophores. To make a photo-electrochemical conversion layer for photocell or biosensor, we deposited PRU liposomes on a solid substrate by electrodeposition. The orientation angle between the molecular axis of PRUs and the disk normal was 2.48 for the deposited PRU liposomes, compared with 20.58 for purified chromatophores deposited. This demonstrates that the vertical alignment of protein molecules with the disk normal was superior for electrodeposited protein molecules immobilized using PRU liposomes compared with those using purified chromatophores. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Rhodopseudomonas Õiridis; Photo-reaction unit; Immobilization; Orientation angle
1. Introduction Purple nonsulfur photosynthetic bacteria contain two types of LH protein complexes. The first type, LH1, surrounds the RC and these make a large protein complex, called a PRU. The second type, LH2, is associated with the PRU and is present in variable amounts in chromatophore membranes. The photosynthetic bacterium Rps. Õiridis we used has only LH1-type protein complexes w1x. The RCs of purple bacteria are widely used to study the key mechanism of photosynthesis: charge separation in BChl b dimer and subsequent electron transfer to primary quinone. LH protein complex binds around RC to transfer the light energy from antenna pigments in LH protein complex to BChl b dimer in RC. We previously made a photocell w2,3x and herbicide
)
Corresponding author. Tel.: q81-298-478883; Fax: q81-298474165; E-mail: [email protected]
sensor w4,5x from a combination of chromatophores, PRUs, and RCs. LH proteins have many chromophores, and the orientation of the Q Y moments of the chromophores are closer to the perpendicular than Q X moments w6x. The cross-section of the absorption moment is maximized for the incident light parallel to the molecular axis. In the chromatophore membranes, RCs transfer electrons across a lipid membrane vertically and unidirectionally. To facilitate this transfer, it is therefore critical to immobilize and manipulate photosynthetic proteins at a desired orientation on a substrate. In this study, first we reconstituted PRUs into liposomes and immobilized PRU liposomes on the solid substrate. The electrodeposited protein molecules immobilized using PRU liposomes were more vertical to the substrate than those immobilized using purified chromatophores. The absorption moments were parallel to the substrate and could therefore absorb effectively with directivity. We then demonstrated PRU liposomes as adequate materials for immobilization by electrodeposition.
0928-4931r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 9 8 . 0 0 0 6 4 - 2
286
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
2. Materials and methods 2.1. Preparation of PRU Chromatophores from Rps. Õiridis were prepared as described by Ajiki et al. w2x. The A 1015 Žabsorbance at 1015 nm. of a chromatophore suspension was measured, and a portion of suspension was mixed with the appropriate amount of Triton X-100 Žfrom a 20% stock solution. and salt to yield a final solution volume of 2 ml that contained A1015 s 150, 5% Žwrv. Triton X-100, and 1 M salt in 10 mM sodium phosphate buffer ŽpH 7.0.. After the solution was incubated for 30 min, it was centrifuged for 15 min at 260,000 = g, and the supernatant was applied to a Sepharose CL-6B ŽPharmacia, Sweden. gel filtration column Ži.d. 2.5 = 100 cm.. The column was equilibrated with a solution of 0.1% Triton X-100, 150 mM LiCl, and 0.02% NaN3 in 10 mM sodium acetate buffer ŽpH 6.0., and then PRUs were eluted with the same solution. All of the above procedures in the PRU preparation were done under dark conditions and at 48C. The PRU fractions were pooled and then concentrated by ultrafiltration ŽDIAFLO PM30, Amicon Div., USA.. The detergent was changed from Triton X-100 to CHAPS-acetate Ž0.1% CHAPS, 150 mM LiCl, and 0.02% NaN3 in 10 mM sodium acetate, pH 6.0. for dialysis. PRU sediments were obtained by centrifuging the PRU solution concentrate for 150 min at 417,000 = g. After the supernatant was removed, the PRU sediments were resuspended in CHAPS-acetate. 2.2. SDS-PAGE SDS-PAGE analysis was done according to Laemmli w7x with slight modification. A 12.5–20% gradient gel was used as separation gel. After the electrophoresis, the gel was stained with Commassie Brilliant Blue G-250. 2.3. Analysis of chromophores Chromophores of the PRUs were analyzed as described by Hara et al. w3x after extraction with acetonermethanol Ž7r2 vrv.. The absorbance was measured at 790 nm, and the BChl b concentration was determined using an extinction coefficient of 122 mMy1 cmy1 reported by Garcia et al. w8x. For the extinction coefficients for the carotenoids, we used the values given by Malhorta et al. w9x: 169 mMy1 cmy1 at 436 nm for neurosporene and its dihydro derivative, and 185 mMy1 cmy1 at 468 nm for lycopene and its dihydro derivative. 2.4. Determination of protein concentration Protein concentration was determined by the method of Lowry et al. w10x. When Triton X-100 was present in the
solution, 0.5% SDS was added to prevent the interference of color development described by Dulley and Grieve w11x. Bovine serum albumin was used as the protein standard. 2.5. Preparation of stock lipid solution For reconstitution into liposomes, we prepared a stock lipid solution as follows. Phosphatidylethanolamine ŽPE; from E. coli, Type IX, Sigma, USA. was used as a phospholipid. Acetone-washed PE Ž40 mg. was dispersed in 4 ml of 50 mM K-HEPES ŽpH 7.8. containing 0.5% deoxycholate, and then sonicated to clarity on ice under a constant stream of nitrogen gas using a probe-type sonicator ŽSONIFIER 450, Branson, USA.. The lipid solution was stored in 1-ml aliquots in a deep freezer Žy808C.. Before use, this stock lipid solution was thawed to room temperature. 2.6. Reconstitution of PRU into liposomes PRUs were reconstituted into liposomes by a dialysis method as described by Molenaar et al. w12x and Crielaard et al. w13,14x with slight modification. PRU solution was added to a PE stock solution to yield a molecular ratio of phospholipids to PRU of 1000:1. After mixing, the solution was dialyzed at 48C for 16–20 h against a 250-fold volume of 10 mM K-HEPES ŽpH 7.8., during which the K-HEPES buffer was changed three times. After dialysis, the liposomes were centrifuged for 10 min at 81,000 = g and resuspended in 5 mM sodium phosphate ŽpH 7.0. by sonication with the probe-type sonicator. The yield of reconstitution was determined by comparing the adsorption spectrum of the supernatant after centrifugation with that of the resuspended solution before centrifugation. 2.7. Electrodeposition of PRU liposomes and chromatophores PRU liposomes and chromatophores were immobilized on a solid substrate electrode by electrodeposition as described by Ajiki et al. w2x. Indium–tin–oxide ŽITO. coated glass ŽGeomatec, Yokohama. was used for the optically transparent substrate electrode Ž2.5 = 25 = 1.1 mm.. Chromatophores were purified for electrodeposition by a sucrose density gradient as described by Jacob and Miller w1x with slight modification. Chromatophores were overlaid onto a discontinuous gradient, which was single layers of 0.5, 1.0, 1.5, and 2.0 M sucrose solutions dissolved in a 10 mM sodium phosphate ŽpH 7.0.. The gradient was centrifuged for 150 min at 417,000 = g, resulting in a purified chromatophore fraction collecting at the interface of the 1.0 and 1.5 M sucrose steps in the
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
287
gradient. The purified chromatophores were washed twice with 5 mM sodium phosphate and then resuspended in the same phosphate buffer adjusted to A1015 s 100. 2.8. Linear dichroism measurement of LH on a solid substrate We determined the orientation by measuring the linear dichroism ŽLD. of chromophores as described by Jacob and Miller w1x, Breton et al. w15x, Nabedryk and Breton w16x, and Breton w17x. The extent of orientation of the PRUs or chromatophores deposited on the ITO electrode was monitored by measuring the LD of the Q Y transitions of BChl b in the LH protein subunits Žat 1006 nm for PRUs or at 1015 nm for chromatophores.. The LD spectra were recorded using a spectrophotometer UV-3101PC ŽShimadzu, Kyoto. that had a polarizer. The light beam used in this measurement was polarized either perpendicular to the normal to the disk plane Žto record A 0 . or parallel to the plane of incidence Žto record A 90 .. Polarized absorption spectra were measured by tilting the sample to 608 with respect to the direction of the beam. Adsorption spectra of the unpolarized light Žto record A N . were recorded without tilting the sample.
3. Results 3.1. Solubilization of chromatophores We studied the effects of salt concentration and type ŽLiCl, NaCl, and KCl. on the solubilization of protein from chromatophores. The yield of solubilization using each salt was estimated from the protein concentration, because the absorption spectrum and SDS-PAGE analysis showed that the solubilized proteins had the same polypeptide composition for all salts. The initial protein concentration was about 12.7 mgrml. KCl was more effective than the other salts in solubilizing PRU ŽFig. 1.. Compared with the protein solubilization in the absence of salt, the addi-
Fig. 2. Absorption spectrum of PRU. Absorption spectrum of ŽŽA. dotted line. native chromatophore, ŽŽB. dashed line. isolated PRU solution, and ŽŽC. solid line. PRU liposomes solution. The peak around 1015 nm in the chromatophore spectrum is slightly shifted to about 1000 nm in the PRU spectrum. The peak around 680 nm in the PRU fraction may be derived from BChl b hydrate.
tion of 1 M KCl increased the solubilized protein concentration by a factor of 2.8. The stability of solubilized PRU was also affected by the salt type and by the pH of the buffer Ždata not shown.. PRU was more stable in buffer that contained LiCl than in those containing the other salts. We therefore used KCl for the solubilization and LiCl was for the elution. 3.2. Preparation of PRU The spectra of the chromatophores and the pooled PRUs ŽFig. 2A and B, respectively. show that the peaks derived from carotenoids and BChl b in PRUs are similar to those from chromatophores. However, the absorbance maximum of BChl b at 1015 nm, derived from BChl b in the LH proteins of chromatophores, was shifted to around 1000 nm in the PRU fraction. BChl b monomer Ž830 nm in RC. and BPhe b are distinguishable in the PRU fraction but not in the chromatophores. The peak around 680 nm in the PRU fraction may be derived from BChl b hydrate. 3.3. Characterization of PRU
Fig. 1. Effect of salt concentration on PRU solubilization. The amount of solubilized protein as a function of salt concentration was determined by the Lowry method. Initial protein concentration was 12.7 mgrml.
The SDS-PAGE analysis of the polypeptide composition of the PRUs ŽFig. 3. shows that the main components of the PRU fraction was RC ŽH, M, L, and Cyt C. and LH proteins Ž a , b, and g ., with trace amounts of other polypeptides. A relative molecular mass Ž Mr . of the PRU Žestimated using HPLC gel filtration. was about 610 kDa in the presence of Triton X-100 Ždata not shown.. For determining the PRU concentration, we used an extinction coefficient of 4.3 mMy1 cmy1 at 1000 nm for BChl b in LH proteins w3x. The calculated number of
288
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290 Table 1 Materials
S
SRC
w Ž8.
Samples
PRU liposomes Chromatophores
y0.327 y0.265
y0.998 y0.816
2.4 20.5
24 21
isolated PRU solution, but recovered to 1006 nm for the PRU liposomes solution. 3.5. LD of immobilized PRUs
Fig. 3. Electrophoresis patterns from SDS-PAGE analysis. Lanes 1, 2, and 3 are chromatophores ŽChr., isolated PRUs, and RCs respectively. Lane 4 is the molecular marker ŽSTD.. The primary components of PRU are RC ŽH, M, L and Cyt C. and LH Ž a , b and g ., with trace amounts of polypeptide.
chromophores per PRU was 27.8 for BChl b and 16.5 for carotenoids. 3.4. Reconstitution of PRUs into liposomes PRUs were reconstituted into PE liposomes using dialysis. The yield of reconstitution was 80% Ždata not shown., which we determined by centrifuging the solution of PRU liposomes. For the chromatophores solution, the peak of the BChl b Q Y moment for LH ŽFig. 2A. occurred at 1015 nm. This peak was shifted to about 1000 nm for the
We measured the LD of immobilized PRUs on ITO substrates to evaluate an alignment of protein molecules. In a quantitative analysis of the orientation distribution of transition moments, Breton reported that for the transition moment M Žcorresponding to one absorption band. oriented with respect to the normal N to the disk, the orientation distribution can be characterized by an order parameter S: Ss
1 y 3 cos 2w
Ž 1.
2
where w is the angle of the orientation distribution between N and the transition moments measured M ŽFig. 4.. The LD of polarized light and the absorbance of unpolarized light is A 0 y A 90
1 s
AN
2'3
=
3S 1qS
.
Ž 2.
Here, S is S s SRC = S LH
Ž 3.
where SRC is the distribution of the two-fold symmetry axis of RC with respect to N and S LH is the distribution of the Q Y transition moments of antennae BChl b in LH proteins. The angle of the Q Y transition moments is 208 with respect to the membrane plane w1x, thereby making it 708 with respect to the two-fold symmetry axis of RC. By Eq. Ž1., therefore the calculated S LH is 0.325. For PRU liposomes electrodeposited on ITO substrates, the measured S was y0.327 Ž N s 24., and the calculated SRC was y1.008. From Eq. Ž1., the range of S is from y1.0 to 0.5. When the angle of this Q Y transition moment was changed from 708 to 70.28 with respect to the two-fold symmetry axis of RC, the calculated SRC is y0.998 and w is 2.48. The following Table 1 lists the results for the measured S and calculated SRC for the electrodeposited PRU liposomes and chromatophores.
4. Discussion Fig. 4. Orientation angle of PRU. Model for the orientation of the transition moment, M. Transition moments of the Q Y band for BChl b in LH make an angle w with the disk normal N.
We previously constructed photoelectric devices and herbicide sensor using chromatophores, PRUs, and RCs.
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
To immobilize photosynthetic proteins on a solid substrate, we tried several methods for controlling the molecular orientation; Langmuir–Blodgett ŽLB. film method, electrodeposition, and the avidin–biotin method w21x. Manipulation and immobilization of photosynthetic proteins at a specific orientation on a substrate is critical because RCs transfer electrons across a lipid membrane vertically and unidirectionally. Our results in this study show that the yield of solubilization is improved by the addition of salt along with Triton X-100. Fig. 1 shows that the amount of total solubilized protein increased with increasing salt concentration. This enhancement depended on the type of salt; KCl ) NaCl ) LiCl. This order of enhancement is the same as the order of the ionic radius of each cation. The addition of NaCl helps to solubilize membrane receptors with ionic detergent w18,19x. However, Triton X-100 is a non-ionic detergent. The reason solubilization is affected by the addition of salt may be the effect of the change in ionic strength or the effect of chaotropic ion-like. Further study is needed to clarify the reason. Using gel filtration, we estimated the Mr of the PRU to be about 610 kDa. Considering that an equal weight of detergent is bound to the membrane protein and that the molecular weight of PRU was estimated by Hara et al. to be about 368 kDa w3x, PRU may be monodispersed in the detergent micelle. Using the extinction coefficients of BChl b and PRU, we estimated the number of BChl b and carotenoid molecules per PRU to be 27.8 and 16.5, respectively. Stark et al. w20x reported that twelve subunits of LH protein, where each subunit has three polypeptides Ž a , b and g ., bind around RC and each subunit contains two BChl b molecules. Therefore, one unit of PRU has 28 BChl b molecules: 4 in the RCs and 24 in the LH proteins. Assuming one carotenoid molecule is contained in the RC and twelve molecules are contained in the LH proteins, the expected number of chromophores per PRU is 28 molecules of BChl b and 13 molecules of carotenoids. The slight peak shift in the isolated PRUs solution around 1000 nm suggests that a slight hydrophobicity or conformational change in the LH proteins occurred by the binding of the detergent rather than the lipid bilayer to the hydrophobic region of PRU or by denaturation of the protein due to the solubilization. This peak shift was not recovered enough by the reconstitution into liposomes. The LH of PRU was distorted by the solubilization, and the peak of BChl b in the LH was shifted to 1006 nm. In the LD analysis, this distortion was taken into account in the calculations. Electrodeposited protein molecules immobilized using PRU liposomes had better vertical alignment compared with those using purified chromatophores. Jacob and Miller w1x reported that the lipids protein ratio of chromatophores is 0.56 Žmg lipidsrmg proteins.. The lipids protein ratio of the PRU liposomes that we prepared was about 1.9 Žmg
289
lipidsrmg proteins.. Compared with chromatophores, PRU liposomes are a relatively ‘soft material’. In this study, we achieved vertical immobilization of PRU by combining a soft material and an electric field.
5. Conclusion Photosynthetic proteins have many chromophores, and the absorption moments Žespecially Q Y moments. are oriented perpendicular to the molecular axis. The crosssection of each absorption moment is maximized for the incident light parallel to the molecular axis. We demonstrated that the vertical alignment of protein molecules with the disk normal was superior for electrodeposited protein molecules immobilized using PRU liposomes compared with those using purified chromatophores. When PRU liposomes were used, the absorption moments were parallel to the substrate and therefore could absorb effectively with directivity. We demonstrated PRU liposomes as adequate materials when immobilization is done by electrodeposition.
Acknowledgements This work was supported in part by the project ‘Research and Development of Protein Molecular Assembly’, which is supported by an R & D Project of Basic Technology for Future Industry, under the Agency of Industrial Science and Technology ŽAIST., Ministry of International Trade and Industry ŽMITI.. We express our thanks to our colleagues at Stanley: Mr. Masami Kumei and Mr. Yoshiaki Yasuda for helpful discussions, and Mr. Yasuhito Ikeda for technical assistance.
References w1x J.S. Jacob, K.R. Miller, Arch. Biochem. Biophys. 223 Ž1983. 282. w2x S. Ajiki, H. Toyotama, M. Hara, J. Miyake, Bioelectrochem. Bioenerg. 43 Ž1997. 71. w3x M. Hara, K. Namba, Y. Hirata, T. Majima, S. Kawamura, Y. Asada, J. Miyake, Plant Cell Physiol. 31 Ž1990. 951. w4x T. Tamura, A. Sato, M. Hara, Y. Asada, J. Miyake, Biotechnol. Tech. 8 Ž1994. 615. w5x H. Peters, C. Schmidt-Dannert, R.D. Schmid, Mater. Sci. Eng. C 4 Ž1997. 227. w6x G. Paillotin, A. Vermeglio, J. Breton, Biochim. Biophys. Acta 545 Ž1979. 249. w7x U.K. Laemmli, Nature 227 Ž1970. 680. w8x A. Garcia, L.P. Vernon, B. Ke, H. Mollenhauer, Biochemistry 7 Ž1968. 326. w9x H.C. Malhorta, G. Britton, T.W. Goodwin, Int. J. Vit. Res. 40 Ž1970. 315. w10x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 Ž1951. 265. w11x J.R. Dulley, P.A. Grieve, Anal. Biochem. 64 Ž1975. 136.
290
S. Ajiki et al.r Materials Science and Engineering C 6 (1998) 285–290
w12x D. Molenaar, W. Crielaad, K.J. Hellingwerf, Biochemistry 27 Ž1988. 2014. w13x W. Crielaad, K.J. Hellingwerf, W.N. Konings, Biochim. Biophys. Acta 973 Ž1989. 205. w14x W. Crielaad, F. van Mouric, R. van Grondelle, W.N. Konings, K.J. Hellingwerf, Biochim. Biophys. Acta 1100 Ž1992. 9. w15x J. Breton, M. Michel-Villaz, G. Paillotin, Biochim. Biophys. Acta 314 Ž1973. 42.
w16x E. Nabedryk, J. Breton, Biochim. Biophys. Acta 635 Ž1981. 515. w17x J. Breton, Biochim. Biophys. Acta 810 Ž1985. 235. w18x R.D. Howells, T.L. Gioannini, J.M. Hiller, E.J. Simon, J. Pharmacol. Exp. Ther. 222 Ž1982. 629. w19x M. Maruyama, H. Sugino, K. Akita, H. Hatanaka, Brain Res. 401 Ž1987. 14. w20x W. Stark, F. Jay, K. Muehlethaler, Arch. Microbiol. 146 Ž1986. 130. w21x J. Miyake, M. Hara, Mater. Sci. Eng. C 4 Ž1997. 213.