Modalizadores en la trinchera

The peridinin chlorophyll protein (PCP) is an unique light-harvesting complex comprised of tightly clustered Chlaand Per pigments with an exceptionally high carotenoid-Chl ratio of 4:1. The highly modified Per features an unusually long-lived S₁-state. These properties render PCP a challenge to understand pigment interactions and excitation energy transfer between Per and Chl as well as among the Chls. In recent years these issues have been addressed by many experimental and theoretical studies. Figure3.10summarises the current understanding of energy transfer pathways in PCP that have been gained from these observations. Excitation transfer from Per to Chl proceeds predominantly via the relatively long-lived S₁state as donor and reaches high efficiency of ∼90-100 %. Research on PCP has been aided using N-PCPs that have been reconstituted with different Chls. Per-Chl and Chl-Chl energy transfer could be studied in a structurally well-known situation that is relatively simple as compared to other photosynthetic systems. In this context, it has been highly advantageous to study PCP containing two different, spectrally separated Chls.

Experimental studies, though extensive, have only been carried out at the ensemble level. In this thesis, the first investigations on PCP using single molecule spectroscopy are presented.

3.6 Conclusion 41

Figure 3.10: Left: Scheme of energy levels and energy transfer pathways between Per and Chl

ain a PCP monomer. Intramolecular relaxation processes are denoted by wavy arrows, while the dashed arrow represents the long-lived Chlafluorescence. Solid arrows represent main energy transfer channels. The dotted arrows represent possible minor energy transfer channel involving higher vibrational levels of the S_1/ICTstate and the S₂–S₂energy transfer involving the Per with blue-shifted absorption spectrum. Excitation is shown as a double arrow. All processes are labelled by the corresponding time constant. From[84]. Right: Energy transfer in a single N- PCP cluster. Shown are the pigments Per 611-614 and Chl 601 colour-coded with their respective excitation wavelength (Table3.2).

SMS removes ensemble averaging and, as will be discussed, allows for monitoring interactions of single weakly coupled fluorescing Chls with neighbouring pigments and their local protein environment. The efficient Per-Chl energy transfer is utilised as an excitation channel for the spectroscopic investigations. The availability of reconstituted homo- and hetero-chlorophyllous N-PCP allows to follow pigment interactions as a function of structure.

CHAPTER

4

Materials and Methods

This chapter describes the sample preparation and experimental procedures that have been applied in the course of spectroscopic investigations of the light-harvesting complex PCP. Section4.1 presents the protocol used for biochemical reconstitution of PCP with various Chls, which results in complexes containing either two identical Chls (homo-chlorophyllous PCP) or two different Chls (hetero-chlorophyllous PCP). The sample preparation method for the optical experiments is given in Section4.2which is followed by a description of the experimental setups for ensemble and single-molecule-spectroscopies in Sections4.3and4.4, respectively. The experimental approach presented in Section4.5adopts the efficient energy transfer between Pers and Chls in PCP as an excitation channel.

4.1 Reconstitution of PCP

PCP can be reconstituted from N-domain coding region apoproteins mixed with pigments extracted from native PCP[94,95]. The possibility of reconstituting PCP complexes with Chls or mixtures of Chls provided a valuable tool to systematically study energy transfer between its pigments as a distinct function of structure. This work was conducted in close collaboration with the group of Prof. H. Scheer (Dept. of Biology, LMU).

4.1.1 Extraction and Purication of N-domain Apoprotein

In a first step N-domain apoprotein was prepared from a 1l culture ofE. coli JM109 on a pND707 plasmid. The culture was grown in a fermenter at 37◦C to an optical density of 0.8 at 600 nm, at which time protein expression was induced by rapidly increasing the temperature to 43◦C. After 3 h at 43◦C, cells were harvested (5000 g, 30 min) by centrifugation, resuspended in 10 ml of lysis buffer (50 mM Tris 1 mM EDTA pH 8.0 + 25 % Sucrose) and lysed by four passages through a French Press (15000 psi). The centrifugation (31000 g, 15 min) of the lysates resulted in pellating of recombinant inclusion bodies. In order to remove the

44 4 Materials and Methods

contaminants from the inclusion body preparation, initial pellets were resuspended in lysis buffer and centrifugated at 12000 g for 15 min. Three cycles and centrifugation (10000 g, 15 min) were then performed in Tris buffer (50 mM, pH 8) containing EDTA (1 mM). Following the addition of 4 ml of 50 mM Tris, 1 mM EDTA at pH 8.0, the inclusion bodies were extracted on boiling water for 20 min. N-domain apoprotein was harvested by centrifugation at 33000 g for 30 min and collected as supernatant. Purity and the size of the apoprotein were tested by SDS-PAGE with SDS-7 (Sigma) as the marker. The apoprotein was charaterised by circular dichroism between 190 - 300 nm (see Chapter 5 and 6). From the initial 1 l culture a concentration of about 1.986 mg ml−1 of N-domain apoprotein (_∼16 kDa) could be collected.

4.1.2 Materials for Reconstitution

Trimeric PCP was isolated fromA. carterae[7]. Per was extracted from thylakoid membranes ofA. carteraeas previously published in[72]. Chlawas extracted from spraydriedSpirulina geitleri, and Chlbwas extracted from frozen spinach and purified over DEAE cellulose[111], acChlawas synthesized from bacterio-chlorophyll (BChl)a(fromRhodobacter sphaeroides

(R26)) and purified by Sepharose CL-6B chromatography[112].

4.1.3 Homo- and Hetero-Chlorophyllous Reconstitution

Reconstitution followed the protocol described in[94]. 200 - 400µg of N-domain apoprotein of PCP (0.32 mg ml−1 or 1.99 mg ml−1) in 50 mM Tris 1 mM EDTA pH 8.0 was combined with tricine buffer (25 mM, pH 7.6) containing KCl (10 mM) and adjusted to achieve a total volume of reconstitution equal to 1000µl. Following the addition of peridinin (12 nmol) and chlorophyll mixtures in 150 µl ethanol, 1 ml of the mixture was incubated at 4 ◦C for 24 hours. The homo-chlorophyllous mixtures contained a total of 3 nmol of either Chl a, Chl

b or acChla. The hetero-chlorophyllous mixtures contained binary mixtures of these Chls that were added up to yield 3 nmol. The different combination ratios of the Chl pigments were 4:1, 2:1, 1:1, 1:2 and 1:4. The crude reconstitution product was loaded on a small Sephadex G-25 column (5x25 mm) and equilibrated with tricine buffer (25 mM, pH 7.6) containing KCl (10 mM) and eluted with the same buffer. Then it was bounded to a column of DEAE Trisacryl equilibrated with tricine buffer (25 mM, pH 7.6) containing KCl (10 mM) and removed with tricine buffer (25 mM, pH 7.6) containing KCl (10 mM), NaCl step gradient (100, 200, and 500 mM). Fractions containing the reconstituted complex (100 mM NaCl) were dialysed against the starting buffer.

The nomenclature used in the text is as follows: homo-chlorophyllous N-PCP is denoted for example as (Chla)2-N-PCP for N-PCP reconstituted with Chla, and hetero-chlorophyllous

N-PCP is denoted for example as (Chla/b)-N-PCP for N-PCP reconstituted with both Chla