Pigment Protein Complexes

The first pigment—protein complexes which were reconstituted are the major LHCII of higher plants and the core LHC

(LH1) of photosynthetic bacteria. In these initial experiments, authentic proteins isolated from LH1 (64) and total thylakoid membrane proteins (72) were employed. New possibilities were opened up by the availability of cDNAs for several LHC-apoproteins of different origins, which allowed the use of bacterially overexpressed LHC proteins in reconstitution experiments (17,68). Since bacterially expressed proteins can easily be mutated, the introduction of various structural alterations into recombinant pigment—protein complexes was facilitated. Using C and N terminally truncated apoproteins, the significance of these protein domains for the formation of LHCII could be identified (18,67). Later, reconstitution of different LHCs of higher plants, CP29 (35), CP26 (82), and CP24 (62), as well as LHCI-730 (86) and LHCI-680a (Schmid and Paulsen, unpublished) were accomplished. Additionally, an LHCI (LhcaR1) of the red alga Porphyridium cruentum (32), a LHC of the green alga Chlorella fusca, and a Chl a,b,c-containing complex of the prasino-phyte Mantoniella squamata were successfully reconstituted (54). Recently, the peripheral LHC (LH2) of a purple bacterium has also been reconstituted (92).

Moreover, it was shown that it is not only possible to reconstitute monomeric LHC but also the oligomeric form of the major LHCII complex and of LHCI-730 (41,79,86).

3.2. Comparison of Reconstitution Procedures

All the LHCII reconstitution experiments until 1992 were performed by the original freeze-thaw method (72). Later, a new method, based on detergent exchange was developed (66), which proved to be very powerful as it has allowed reconstitution of the more labile complexes in recent years. In Table 3, these two methods and their applications are summarized. Both procedures will be given in more detail in section 3.3.2.

The result of reconstitutions of LHCI and LHCII by these two methods is depicted in Figure 1. It is obvious that application of the detergent exchange method yields reconstituted LHCI and LHCII, whereas with the freeze-thaw method, only reconstituted LHCII is obtained.

Additionally, a reconstitution technique was developed, which is based on detergent mixing, which also prompts protein folding and pigment binding. This method allows protein refolding to initiate very quickly and, therefore, facilitates time-resolved spectroscopic measurements (8).

3.3. Reconstitution Procedures

Reconstitutions of plant LHCs usually start from isolated plant pigments and bac-terially expressed apoprotein.

3.3.1. Pigment Isolation

Reconstitution is performed either with a total thylakoid pigment extract or with (a mixture of) individual pigments. Some pigments are commercially available [e.g., Chl a and b, lutein, a- and P-carotene from Sigma (St. Louis, MO, USA); lutein and zeaxanthin from Roth (Germany)] but others are not. Therefore, their isolation is described in the following protocol. All isolation steps should be performed in dim light. As a source for pigment isolation, homogenized whole leaves or thylakoids isolated as in, e.g., Reference 16, can be used. Thylakoids are suspended in a small volume of dilute buffer as 10 mM Tricine-NaOH (pH 7.8) and extracted by addition of acetone to a final concentration of 80%. Proteins are removed by a 10-minute cen-trifugation at 15 000x g. For isolation of total pigment extract or individual pig ments, the supernatant is treated in different ways.

❖ Procedure 1. Total Pigment Extract

1. The pigment solution in acetone is mixed with 0.25 volumes diethyl ether in a separating funnel.

2. To improve phase separation, solid NaCl (e.g., 35 g to 600-mL solution) is added and dissolved by gently moving the funnel. If the lower acetone phase remains colored, the ether extraction should be repeated, adding more NaCl if phase separation is poor.

3. Combine and dry the ether phases, either by the addition of solid NaCl or by placing the ether solution in a -20°C freezer for at least 1 hour. Then ice or NaCl can be removed by filtration through a sintered glass funnel (precooled to -20°C if ice crystals are to be removed).

4. Evaporate the ether in a rotary evaporator or nitrogen stream.

5. Pigments are dissolved in acetone, quantified on the basis of their Chl content (see section 2.2.1), and aliquoted.

Figure 1. Partially denaturing gel electrophoresis of reconstitution mixtures with LHCI- (Lhca4; lanes a and b) or LHCII- (Lhcbl; lanes c and d) apoprotein. The mixtures were subjected to either the freeze-thaw (lanes a and c) or the detergent exchange method (lanes b and d). The resolution of bands with monomeric complexes (M) and free pigments (FP) is visible.

Figure 1. Partially denaturing gel electrophoresis of reconstitution mixtures with LHCI- (Lhca4; lanes a and b) or LHCII- (Lhcbl; lanes c and d) apoprotein. The mixtures were subjected to either the freeze-thaw (lanes a and c) or the detergent exchange method (lanes b and d). The resolution of bands with monomeric complexes (M) and free pigments (FP) is visible.

6. The aliquots are dried in a nitrogen stream and can be stored for months to years at -20°C under nitrogen or argon. For the isolation of individual Chls and carotenoids, the following procedure is useful:

❖ Procedure 2. Isolation of Individual Pigments

1. The acetonic pigment solution is cooled to 0°C.

2. Dioxane is added to give a final concentration of about 15% (vol/vol) (42).

3. To the homogenous solution 0.16 volumes of water is added drop-wise under constant stirring, and the resultant solution is kept on ice for 1 hour without further stirring.

4. Aggregated Chls (as well as pheophytin and P-carotene) are collected by cen-trifugation (15 000 x g, for 10 min), and the pelleted pigments are used for column chromatographic separation of the Chls and P-carotene. If the supernatant still contains a substantial amount of the Chl originally present, more water may be added drop-wise, and the additional precipitate collected. If the addition of water is too excessive or too fast, xanthophylls will also aggregate and contaminate the crude Chl preparation. The supernatant is kept for xanthophyll isolation.

5. For the separation of individual Chls (and xanthophylls), a reversed phase C18 material [e.g., 55-105 p-Bonda-pak (Waters, Milford, MA, USA)] with acetone-water mixtures as the mobile phase is suitable. Chromatography can be done either at low pressure or by using an HPLC apparatus. A column volume of at least 4 mL (low pressure development) or 0.8 mL (high pressure processing) is recommended for each milligram of raw pigments applied.

Table 3. Comparison of the Experimental Steps of the Two Most Commonly Used Reconstitution Techniques
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