PhotosynthesisResearch 46: 339-345, 1995. O 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Perfusion chromatography - a new procedure for very rapid isolation of integral photosynthetic membrane proteins M a r g r i t R o o b o l - B 6 z a 1, S u s a n a S h o c h a t 2'3, S t a f f a n E. T j u s 1, A s a H a g m a n 1, P e t e r G a s t z & Bertil A n d e r s s o n 1 IDepartment of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden; 2Department of Biophysics, Huygens Laboratory, State University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; 3Department of Biological Chemistry, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received26 April 1995;acceptedin revisedform8 June 1995

Key words: bacterial reaction centres, chlorophyll-binding proteins, hydrophobic membrane proteins, Photosystem I, Photosystem II

Abstract

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.

Abbreviations: C h l - chlorophyll; L D A O - N,N dimethyldodecylamine-N-oxide;L H C - light-harvesting complex; PS - photosystem; SDS-PAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresis Introduction

During the last 10 years, the field of life sciences experienced significant technological advances. The possibility to analyse, modify and multiply the genetic materiai in combination with techniques for over-expression of gene-products have largely changed the experimental strategies, a development that has also influenced photosynthesis research. Today we know of at least 60 different proteins in the thylakoid membrane and for some of them the structural and functional details are known (Anders-

son and Barber 1994). Still, the functional significance is not known for the majority of the thylakoid proteins. One major reason is that when it comes to the purification and isolation of hydrophobic and integral membrane proteins, despite the technological developments, the methods are still fairly crude and elaborate. The isolation of hydrophobic membrane proteins or protein complexes require solubilization by detergents combined with a specific kind of separation technique. Even though several photosynthetic protein complexes have been isolated by this general experimental strategy (Andersson and Anderson 1985), the fractionation

340

Fig. 1. The POROS beads used for Perfusion chromatography can be 10, 20 and 50 micronsin size dependingon the application. The beads contain two types of pores; through pores and diffuse pores. In the schematicrepresentation,the blackbeadsare shownin a dissection, where the pores are represented as white tunnels. The different mobileflows are depictedby arrows.

methods involve a trial and error optimisation of the appropriate conditions for solubilization, and the subsequent isolation normally requires several hours or even days for completion. In this paper, we will describe how the new method of perfusion chromatography (Regnier 1991) can be applied as a versatile tool for the very rapid isolation of photosynthetic complexes from bacterial and chloroplast membranes.

Basic principles of perfusion chromatography Perfusion chromatography has drastically reduced the time required for separation of soluble proteins (Afeyan et al. 1991) and peptides (Fulton et al. 1992). It takes advantage of a recent development in the use of the chromatographic matrix (Regnier 1991; Fulton et al. 1992). In conventional liquid chromatography, the resolution of protein bands during a separation can be disturbed by intra-particle diffusion since stagnant locations in the mobile phase of the column lead to retardation and band broadening (Afeyan et al. 1990). POROS, the perfusion chromatography matrix (Perseptive Biosystems) is a very high porosity bead, containing two types of pores (Fig. 1) that permits the

mobile phase to pass through the gel matrix without retardation. Each of the two pore types play a specific and different functional role. The 'through pores' range from 6000-8000, g, in diameter and rapidly guide the liquid flow through the interior of the gel matrix (Afeyan et al. 1990; Regnier 1991). Therefore, the through flow can transport solutes to the inside of the particle in a much more efficient maner than is done by the diffusive flow in traditional chromatographic particles. The second type of pores are smaller pores of 800-1500 ~k in diameter that increase the surface area and thus enhance the loading capacity (Fig. 1). These 'diffusive pores' are maximally 1/~m in length (Afeyan et al. 1991). Simultaneously, the pore morphology is determined by the number of diffuse pores in addition to the through pores. The optimal ratio between the two types of pores was found to meet the two important requirements; minimal resistance to mass transfer (large 'through pores') and a high loading capacity (a high number of 'diffusive pores'). Under optimal conditions, it is possible to separate proteins at least ten times faster than by conventional chromatographic methods, combined with a loading capacity of up to four times larger than most other anion exchange matrixes (Afeyan et al. 1990).

Isolation of Photosystem II reaction centres The study of PS II electron transport has been facilitated by the isolation of oxygen-evolving core complexes and reaction centre particles (see Satoh 1993; Seibert 1993). The latter type of preparation contains the D1 and D2 proteins, comprising the reaction centre heterodimer thought to harbour all redox components involved in water-plastoquinone oxidoreduction. In addition the reaction centre preparation contains cytochrome b559 and the psbI (Satoh 1993; Seibert 1993) and psbW (Irrgang et al. 1995) geneproducts. The initial work by Nanba and Satoh (1987) and Barber et al. (1987) was based on Triton X-100 solubilization of spinach or pea thylakoids, respectively, followed by overnight DEAE anion exchange chromatography. Later, several modifications of these preparations, in particular the introduction of octyl glucopyranoside and dodecyl maltoside for solubilization, rendered the isolated reaction centres more stable (Chapman et al. 1988). Fotinou and Ghanotakis (1990) combined the use of these two detergents followed by a two step anion exchange chromatography (Fast flow Q, Pharmacia Biotech). An additional aspect in this

341

Fig. 2. The elution profile during PS II reaction centre isolation by perfusion chromatography. The pure PS II reaction centre complexes are eluted from the column in the third peak within in 6.5 rain of elution. This fraction is indicated by an arrow. The SDS-PAGE protein profile of this fraction is shown (inset). The SDS-PAGE shows the presence of the 1)2 and D1 proteins as well a s the cytochrorae b559 and psbI gene product.

method is the use of LiC104 during both solubilization of the PS II core complexes and the subsequent elution. In the first step the major chlorophyll a/b proteins are removed, and in the second step PS II core proteins like CP 47 and CP 43 are separated from the reaction centre particles. Van Leeuwen et al. (1991) developed the procedure further by introducing MgSO4 in combination with dodecyl maltoside initially, and Triton X100 later, and an anion exchange column (Sepharose Q, Pharmacia Biotech). The overall preparation time in this procedure compared to previous methods was reduced because the chromatographic step for the PS II core complexes required 50 min, and for the PS II reaction centres 90 min. Despite these modifications and improvements in the preparation during recent years, all procedures have so far relied on conventional anion exchange chromatography. By applying anion exchange perfusion chromatography, we have been able to drastically reduce the time required for the isolation of both oxygen-evolving PS II core complexes and PS II reaction centres from spinach thylakoid membranes (Roobol-B6za and Andersson 1995). PS II-enriched membranes were isolated by the method of Ghanotakis et al. (1984), and were finally suspended in 20 mM Bis-Tris (pH 6.5), 20 mM MgCI2, 5 mM CaC12, 10 mM MgSO4, 0.4 M sucrose and 0.03% (w/v) dodecyl maltoside (Sigma), to a final concentration of 2 mg Chl/ml according to van Leeuwen et al. (1991 ). After centrifugation, the supernatant was load-

ed on a POROS Q anion exchange column (PerSeptive Biosystems) attached to a FPLC system (Pharmacia Biotech). The column was then washed with equilibration buffer containing 5 mM MgSO4 at a flow rate of 1 ml min-1. After only 10 to 15 rain, the PS II core complexes were eluted from the column with 250 mM MgSOa(Roobol-B6za and Andersson 1995). These PS II core complexes were very pure as judged by SDS-PAGE and showed high rates of oxygen-evolving activity. For further fractionation into PS II reaction centres, the obtained material was desalted using an ultra filtration cell (Amicon, cut-off 30 kDa) and concentrated to a final chlorophyll concentration of 500/~g Chl/ml. It was then incubated with an equal volume of 20 mM Bis-Tris (pH 6.0), 50 mM dodecyl maltoside (2,5%), 1% Triton X-100 and 4 M LiC104 (equilibration buffer). After 15 min solubilization in the dark and on ice, the solution was quickly desalted on a PD-10 column (Sephadex G-25, Pharmacia Biotech) and then transferred to the POROS Q column (1 ml wet volume) connected to the FPLC system. The sample was loaded and washed with 2 column volumes (2 ml) of the equilibration buffer, after which the LiC104 concentration was increased to 90 mM to start the elution. After 4 ml of 90 mM LiC104 the concentration was further increased to 125 mM (4 ml), than 200 mM LiC104 (4 ml) and eventually the column was eluted with 6 ml of 300 mM of the salt (Fig. 2). The elution profile reveals five different peaks (Fig. 2). The major contaminants, as the LHC II, CP 43 and the CP 47, were eluted in peak one, two and four in different combinations with other PS II core polypeptides. The PS II reaction centres were recovered in peak three. The purity was judged by absorption spectroscopy (417 nm/435 n m > 1.2) as well as by SDS-PAGE, which shows both the D1 and D2 protein in two distinct bands and the cytochrome b559 andpsbI gene product together in the low molecular mass region (see inset in Fig. 2). Peak three in the elution profile shows a shoulder which was assigned to a CP 47-reaction centre complex. Notably, the PS II reaction centre fraction could be collected as soon as 6.5 min after loading the solubilized material on the column.

Subfractionation of chlorophyll-binding proteins of Photosystem I Photosystem I (PS I) is a multi-component complex containing a large number of polypeptide subunits

342

Native photosystem I Photosystem I core complex

Lhca2

Z

~I LHC~140

Omin/ mM

YYY}i

..... . . . ...-..,.-.~.?

~_'.$.~L~?.,.".~:..

22 rain/ Lhca3 Lheal+2+4 Lheal+4

Lhea2+3

Fig. 3. Schematic illustration of subfractionation of native PS I complexes by perfusion chromatography. Each arrow represents a specific elution time and salt concentration needed to isolate the corresponding subcomplex.

(Ikeuchi 1992). The reaction centre is composed of two high molecular mass subunits, psaA and psaB, which contain several of the redox components required for the primary photochemistry, as well as a large number of chlorophyll a antenna molecules. In addition, P S I carries its own chlorophyll a/b antenna designated LHC I. This complex is known to be composed of 4 proteins; 21,5 kDa Lhcal, 23 kDa Lhca2, 24 kDa Lhca3 and 21 kDa Lhca4 (see Jansson 1994). By solubilization of native P S I complexes from pea with the non-ionic detergent dodecyl maltoside (maltoside:chlorophyll = 3:1, w/w) and the charged Zwittergent (Zwittergent:chlorophyll = 4:1, w/w), the PS I core complex and LHC I were first separated and isolated following a 20 h sucrose gradient ultracentrifugation (Haworth et al. 1983). Later, Lam et al. (1984) subfractionated LHC I, prepared as in Haworth et al. (1983), into two chlorophyll a/b binding subcomplexes designated LHC PIa and LHC PIb. This separation was achieved by an additional 30 hour sucrose gradient ultracentrifugation of the original LHC I fraction, yielding two closely migrating green bands. The lighter LHC PIa and heavier LHC PIb showed maximal 77 K fluorescence at 680 nm and 730 nm, respectively. These fractions have accordingly been designated LHC 1-680 and LHC 1-730 (Bassi and Simpson 1987). LHC 1-680 is composed of the Lhca2+3 proteins, and LHC 1-730 by the Lhcal+4 proteins. Ikeuchi et al. (1991) and Knoet-

zel et al. (1992) have recently gone further and subfractionated LHC 1-680 into single pigment-binding polypeptides, called LHC 1-680A (Lhca3) and LHC 1680B (Lhca2), by mild SDS-PAGE or sucrose gradient ultracentrifugation. In an attempt to fractionate PS I from spinach by perfusion chromatography, native PS I complexes were solubilized by dodecyl maltoside and Zwittergent and loaded onto an anion-exchange POROS Q column (Tjus et al. 1995). Elution by a NaC1 gradient (0-400 raM) yielded P S I core complexes containing the reaction centre in only 30 min after the onset of solubilization (Fig. 3). This time should be compared to the 20 hours required when previous methods were employed, based upon sucrose gradient centrifugation (Haworth et al. 1983). The isolated P S I core complex showed very high electron transport activity (1535 #mol 02 consumed mg chl-l.h - l)when using methylviologen as an electron acceptor. It was clearly devoid of chlorophyll b and showed a typical 77 K fluorescence maximum at 724 nm (Haworth et al. 1983; P~lsson et al. 1995). Furthermore, its fluorescence quantum yield was notably as low as 0.4%, demonstrating the isolation of a highly intact P S I core complex. The preparation was shown by SDS-PAGE to retain all the known PS I core polypeptides (Tjus et al. 1995). The perfusion chromatography also allowed a very rapid and efficient subfractionation of LHC I. Loading the solubilized native PS I onto the POROS Q column several LHC I subfractions were eluted; Lhca2+3 (LHC 1-680), Lhcal+4 (LHC 1-730) and Lhca2 (Fig. 3). LHC 1-680 and LHCI-730 were obtained within 20 min after solubilization of the native P S I (Tjus et al. 1995), which is a significant improvement as compared to the 50 h required using the sucrose gradient centrifugation procedure (Lam et al. 1984). In addition, after a somewhat milder solubilization, Lhca3 and Lhcal+2+4 fractions were obtained only 10 min after loading the samples onto the column (Tjus et al. 1995), enabling the very first isolation of these subcomplexes of LHC I in a soluble form and without the use of SDS. The purity and properties of the isolated LHC I subfractionations were assayed by absorption and fluorescence spectroscopy as well as by SDS-PAGE (Tjus et al. 1995).

343 Isolation of reaction centres from Rhodobacter sphaeroides The reaction centre protein complex of photosynthetic purple bacteria is embedded in the cytoplasmic membrane, and is surrounded by the two lightharvesting antenna complexes termed B870 and B800850 according to their absorption maxima. The reaction centre of Rhodobacter sphaeroides consists of three protein subunits, designated H (heavy), M (medium) and L (light), according to their apparent molecular mass as determined by SDS-PAGE (Okamura et al. 1974). The three dimensional structure of the reaction centres of Rhodopsedomonas viridis (Michel et al. 1986) and ofRhodobacter sphaeroides (Allen et al. 1987), elucidated by means of X-ray diffraction, show that two homologous transmembrane protein subunits, L and M, harbour all the redox components required for primary photochemistry. The H subunit is located at the cytoplasmic side of the membrane, with one helix extending into the membrane. All the accepted procedures used for isolation of reaction centres from purple bacteria include their extraction from chromatophores by incubation with a suitable detergent and further purification by ammonium sulphate fractionation and ion exchange chromatography (Gingras 1978). Several variations of this general approach have been developed for the different purple bacteria and different strains of the same bacteria. Even for the same strain, when cells are grown under different light conditions and therefore contain a different pigment composition, other conditions for the extraction of the reaction centres are needed. The detergent that has been most useful is the detergent dodecyl dimethylamine oxide (LDAO), the use of which was introduced by Feher (1971) and by Clayton and Wang (1971 ), for the extraction of the photochemical reaction centre from the R-26 strain of Rhodobacter sphaeroides, and later also used with wild type cells (Jolchine and Reiss-Husson 1974). In our experience, the extraction of the wild type of Rhodobacter sphaeroides (strain 2.41 grown in light) gives the highest yield when incubating the chromatophores at a concentration of A 850 nm = 50 in 100 mM phosphate buffer pH 7.5 containing 0.3% LDAO for 30 min at 30 °C. However in several instances, like in the case of wild-type cells grown at low light or mutants grown in the dark (Shochat et al. 1994), the reaction centres were not extracted after a single incubation with detergent, but a double and sometimes even a triple incubation of the chromatophores in the presence of

Fig. 4. The elution profile for the isolation of bacterial reaction centres from Rhodobacter sphaeroides. The pure reaction centres are eluted from the column in the second fraction. The purity is illustrated by the AEso/As02 = l.l - 1.3 and by the SDS-PAGE protein profile (inset) revealing the presence of the L, M and H subunits only.

0.25% LDAO was required to extract a high yield of reaction centres. Similar observations have been made for the green strain of Rhodobacter sphaeroides (Gray et al. 1990). Moreover, we have found that there is always a co-extraction of light-harvesting complexes from the chromatophores, which represent the main impurity of the preparation. The extracted reaction centres are further purified by ammonium sulphate fractionation (30%). The floating pellet obtained is resuspended in Tris-HCl buffer (pH 8.0), containing 0.1% LDAO. The final purification of the extracted reaction centres involves anion exchange chromatography to separate the reaction centres from the contaminants by elution with a continuous 100-300 mM NaC1 gradient, after washing with the resuspension buffer. We have found that using materials like DEAE Sephacel or Q Sepharose, the elution peaks of the reaction centres and the light-harvesting complex overlap and the yield of pure reaction centres is low, as a consequence several sequential separations were needed for satisfactory fractionation. Due to the intrinsic flow properties of these traditional chromatographic material and the need for repetitive separations, the isolation required several hours for successful completion. Therefore, perfusion chromatography was applied to isolate reaction centres, after LDAO extraction of chromatophores from Rhodobac-

ter sphaeroides.

344 After loading the extract on to the POROS Q, and subsequent washing with the resuspension buffer, followed by elution with the same NaC1 elution gradient as described above, we were able to obtain two well resolved elution peaks containing reaction centres (Fig. 4). Peak one, at the beginning of the gradient, represents about 20% of the reaction centres, and is not a clean fraction (A280/Aso2 = 2). Peak two is the major peak of pure reaction centres (A280/As02 = 1.1 - !.3), eluting at around 200 mM NaC1 (Fig. 4). As shown by SDS-PAGE, this fraction contains only the L, M and H subunits (inset in Fig. 4). The pure light-harvesting complexes elute at around 400 mM NaCI (not shown). Notably, only one purification step was required to obtain a pure reaction centre preparation.

Future perspectives The examples described above show that perfusion chromatography can be used for separation of hydrophobic protein complexes from photosynthetic membranes. The time needed for the separation is drastically reduced, compared to that required by currently accepted methods, and some of the chromatographic steps require only a few minutes for completion. Such 'instant' separations are not only beneficial in terms of convenience, but perfusion chromatography opens up new possibilities and strategies for membrane protein purification. The very short time of purification would increase the possibilities of obtaining membrane proteins in a more native and functional state including the maintenance of ligands. Moreover, it may allow the isolation of protein complexes trapped in transient metabolic or structural states. The speed of separation will also make it realistic to perform sequential separations, and to combine several physicochemical parameters for optimal separation of heterogeneous mixtures of membrane proteins. Finally, when a separation can be completed within minutes, there is a possibility of 'chromatographic screening' in order to find optimal subfractionation conditions.

Acknowledgements This work was supported by the Swedish Natural Science Research Council and the G6ran Gustafsson Foundation for Research in Natural Sciences and Medicine.

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