Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

Summary

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

Abstract

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

Introduction

Oxygenic photosynthesis is one of the most important chemical reactions on our planet. The conversion of light to chemical energy occurs in the reaction centers of two photosystems, photosystem I (PSI) and photosystem II (PSII)¹ (Figure 1A). PSI is a large, highly conserved multisubunit pigment-protein complex that evolved over 3.5 billion years ago^2,3. This complex, which contains approximately 100 chlorophyll molecules and about 20 carotenoids, facilitates the transfer of electrons across the thylakoid membrane from plastocyanin to ferredoxin acting as the terminal electron acceptor of the photosynthetic electron transport chain^1,4,5(Figure 1B, C). In plants, this light-driven charge separation is the result of light energy transferred from both PSI core antenna pigments and the peripheral antenna pigments of Light Harvesting complex I (LHCI) to the PSI reaction center (Figure 1D). LHCI is a PSI-specific antenna complex within the thylakoid membrane composed of four chlorophyll a/b binding LHCA antenna proteins^6,7.

Figure 1: The photosynthetic electron transport chain and the overall structure of the PSI-LHCI complex. (A) The photosynthetic electron transport chain contains four main membrane-bound photosynthetic complexes and three soluble electron carriers. Electron flow (red arrows) through the transport chain and proton pumping (black arrows) into the lumen are used to create reducing power (NADPH) and produce ATP for carbon fixation^37,38,39,40. Created with Biorender.com. (B) The structure of the plant PSI-LHCI from the lumenal side. PsaA and PsaB are the largest subunits of PSI and comprise the core of the complex. LHCI is the light-harvesting antenna complex associated with PSI and is composed of four antennae, LHCA1-4. (C) The PSI-LHCI complex coordinates over 150 ligands. Shown here are chlorophylls (green), carotenoids (pink), quinones (purple), lipids (orange), and the FeS clusters of the reaction center in yellow/orange. (D) The reaction center of PSI is split into two branches (A and B), starting from P700, the reaction center special chlorophyll pair, going into two accessory chlorophylls (A_-1A/B) followed by another pair of chlorophylls (A_0A/B). These chlorophylls are followed by a phylloquinone (A_1A/B or Q_A/B in some publications) in each branch before joining together at the iron-sulfur cluster F_x followed by two more clusters, F_A and F_B, coordinated by the PsaC subunit. Please click here to view a larger version of this figure.

The first isolation of PSI from plants in 1966 shed light on the differences in light-harvesting pigment content between PSI and PSII, showing that PSI was highly enriched in β-carotene relative to PSII and that cytochromes f and b₆ (part of the cytochrome b₆f complexes) are not tightly bound to PSI but loosely associated within the thylakoid membrane⁸. Nine years later, with partial denaturation of isolated PSI via SDS treatment it was shown that dissociation of small PSI subunits quenched NADP+ photoreduction by PSI, while the P700 signal and most of the chlorophylls remained within the remaining large molecular weight PSI particle, identifying the necessity of some of PSI's small subunits for full biological function and the location of the PSI reaction center⁹. Research into the association between the PSI core and LHCI was first published in the early 1980s, when isolations of different-sized PSI species containing differing ratios of chlorophyll A to P700 were observed, suggesting the association of PSI with a chlorophyll-containing peripheral antenna system^10,11,12,13. However, it wasn't until 2003 that the first crystal structure of the plant PSI was published¹⁴. The crystal structure of the plant PSI-LHCI highlighted the remarkable conservation between the PSI core of plants and cyanobacteria and provided the first picture of chlorophyll arrangement within the plant PSI core and LHCI antenna, furthering the understanding of the energy transfer pathways within the plant PSI-LHCI complex¹⁴. Over the past decade more plant PSI-LHCI structures were determined adding atomic levels details to the structural description of the super-complex^{15,16,17,18,19}.

PSI not only has a quantum efficiency close to one, but boasts the most negative reduction potential in nature^20,21. A complete understanding of PSI-LHCI and its properties is essential for understanding light driven energy transfer and applying bio-inspired solutions to future light harvesting technology. To further this understanding of how PSI-LHCI and its many subunits can achieve such efficient energy conversion, complexes isolated for study must be active and whole. This protocol allows for the gentle purification of the complex in this active state^22,23.

In this method plant tissues are mechanically disrupted and chloroplasts containing the photosynthetic electron transport chain are isolated by centrifugation. The thylakoid membranes are separated after hypotonic chloroplast lysis and are then solubilized using the detergent n-dodecyl-beta-maltoside (β-DDM). The solubilized chlorophyll containing membrane complexes are separated using anion exchange chromatography and PSI-LHCI is further separated using sucrose gradient centrifugation. After removal from the gradient and after characterization by both spectroscopy and using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the complex can be prepared for further experiments. This procedure is used to purify the PSI-LHCI complex from plants without the use of any affinity tags. With minor modifications it can be adapted for preparations of the complex from other organisms, stabilize alternative PSI complexes or other complexes of the photosynthetic electron transport chain. Similar protocols were used to obtain PSI complex suitable for high resolution structural analysis^{23,24,25,26,27,28,29,30}.

Protocol

1. Preparation of thylakoid membranes from spinach leaves

1. Work on ice and avoid exposure to light where possible in this preparation. A low light environment is sufficient if high chlorophyll concentrations are maintained, and care is taken to keep the samples covered as much as possible. Perform all centrifugation steps at 4 °C unless specified otherwise.
2. Remove stems from the spinach leaves (500 g) using scissors, gently press the leaves into a blender and completely cover with ice-cold sucrose, tricine, NaCl (STN) buffer (Table 1). Homogenize plant tissue in STN buffer for 10 s twice (total 20 s), leaving 10 s between pulses to prevent overheating using the pulse setting.
   NOTE: A typical ratio of buffer to leaves is 1 L of STN buffer for 500 g of spinach leaves.
3. Filter cell debris through eight layers of cheesecloth by securing the layers of cheesecloth over a large, chilled beaker or other receptacle and pouring the blended mixture through the cloth. Use a glass rod or spoon to stir the cell debris in the filter to allow lysate to flow through.
4. Pellet chloroplasts from the cell lysate by centrifugation at 1000 x g for 9 min. Resuspend chloroplasts in 1 L of hypotonic buffer (Table 1). To resuspend chloroplasts, use a pipette or swirl chloroplast pellet in buffer and transfer to a tissue grinder or a similar homogenizer at an appropriate size.
   NOTE: Exposure to hypotonic solution and resuspension in a tissue grinder lyses the chloroplasts.
5. Centrifuge for 2 min at 500 x g to remove starch (collected as a white pellet). Gently resuspend any green material that has pelleted back into the supernatant using a pipette or a small paint brush while avoiding starch resuspension as much as possible. Centrifuge the solution at 12,000 x g for 10 min to pellet thylakoid membranes.
6. Resuspend the pelleted membranes in minimal amounts of the high salt resuspension buffer (Table 1), only enough to resuspend the membranes, using a pipette or small paint brush and homogenize using a homogenizer as in step 1.4. Centrifuge at 8000 x g for 10 min.
7. Resuspend as before in minimal amounts of STN2 buffer (Table 1), only enough to resuspend membranes and measure chlorophyll content using a UV-Vis spectrophotometer. Determine chlorophyll concentration by diluting the sample 1/1000 in 80% acetone solution and using the method of Porra et al.³¹.
8. Adjust to a desired chlorophyll concentration, typically 3 mg/mL, by adding STN2 buffer and aliquot evenly into two or three centrifuge tubes. The expected yield from 500 g of leaves is around 200 mg of chlorophyll, but variation based on the state of the starting material is expected. Freeze at -80 °C until ready for membrane solubilization. Membranes frozen at this stage are stable for at least 6 months.

2. Membrane solubilization

1. Solubilize thylakoid membranes with β-DDM. Add enough detergent from a 10% β-DDM stock to achieve a 6:1 β-DDM to chlorophyll ratio. Incubate on ice for 30 min. Gently mix every 5-10 min by slowly inverting the tube several times.
2. Centrifuge at 120,000 x g for 30 min using an ultracentrifuge to remove insoluble material. Discard the pellet and save the supernatant for subsequent steps.

3. Elution using diethylaminoethyl (DEAE) column

1. Prepare the anion exchange column using 1.5 mL bed volume for 1 mg of total chlorophyll in the solubilized sample. Prepare the column and run at 4 °C. Pour the resin into a column secured onto a ring stand. Allow the resin to settle while adding water or column low salt buffer to prevent the column from running dry. Ensure the column is free of bubbles and the top is flat and level.
2. Wash the column using two column volumes (CV) of column low salt buffer (Table 1) and load the supernatant from step 2.2 onto the column. Allow the supernatant to completely run into the column before moving on.
3. Wash the column in one CV of column low salt buffer.
4. Elute using a linear NaCl gradient with the low salt and high salt column buffers (5-250 mM NaCl; Table 1) made in a total volume of six CV (i.e., if the column bed volume is 30 mL, use 90 mL of low salt buffer and 90 mL of high salt buffer).
   1. Fill two beakers with the low and high salt buffers. While dripping low salt buffer into the column, create a salt bridge between the high and low salt buffers using a tube filled with water to gradually mix them. Use a stir bar to ensure that the high salt buffer moving into the low salt buffer is being mixed before it is dripped into the column. This ensures that the NaCl gradient is linear. Begin collecting fractions.
5. Collect 4 mL fractions (for a typical experiment starting with about 35 mg of chlorophyll) and combine the dark green fractions eluting around the last one-third of the gradient (Figure 2A).

4. Polyethylene glycol (PEG) precipitation

1. To the combined chlorophyll fractions, slowly add PEG6000 to a final concentration of 8%. The PEG concentration can vary a bit; if precipitation is not seen after reaching 8%, increase the PEG concentration in 2% increments until precipitation is seen (the solution will turn cloudy).
2. Centrifuge at 3,214 x g for 5 min. Discard the supernatant completely; complete removal of all residual PEG is essential to achieve good solubilization at the next step. Resuspend the green precipitate in 2-5 mL of post-column resuspension buffer (Table 1).
3. To verify that thylakoids have been completely washed of PEG, spin a small aliquot of the resuspended material in a benchtop centrifuge at 18,407 x g at 4 °C for 5 min to ensure the material is soluble. A small green precipitate is acceptable at this stage, but most, if not all, the chlorophyll should remain in the solution. Keep the soluble fraction.

5. Preparation of sucrose gradients

1. Prepare 10%-30% discontinuous sucrose gradients in five layers (10%, 15%, 20%, 25%, and 30% layers) with sucrose gradient buffer (Table 1). Assemble the sucrose gradients by carefully layering sucrose fractions in a polyallomer centrifuge tube, starting with the 30% layer and ending with the 10% layer. Adjust the volume of every layer so that there is enough headspace at the top of the tube to load the desired volume of sample.
2. Load samples from the soluble material from step 4.2 into sucrose gradients. Load enough sample to equal 170 µg of chlorophyll into one tube, and 420 µg of chlorophyll into another to obtain gradients at different concentrations to assess each band's homogeneity (Figure 3A).
   NOTE: The amount of sample added is arbitrary but a range of about 150 to 500 µg of chlorophyll is usually appropriate.
3. Centrifuge the gradients at 100,000 x g for 16 h to separate the different complexes within the sample.

6. Removal of sucrose fractions and PEG precipitation

1. After centrifugation, remove the gradients from the rotor and take pictures of the tubes using a digital camera or cellphone. Isolate the fractions by piercing the tubes at the bottom with a needle and then slowly draining the contents. Collect the different chlorophyll-containing fractions in centrifuge tubes.
   NOTE: The samples can be aliquoted and frozen for subsequent analysis if desired.
2. For some applications removal of sucrose is desirable. To remove sucrose, use a second step of PEG precipitation (or gel filtration). Adjust the NaCl concentration in the PSI-LHCI fraction to 120 mM and add PEG6000 to a final concentration of 10%.
3. Centrifuge the sample in a benchtop centrifuge at 18,407 x g at 4 °C for 5 min. Resuspend the green pellet in 30 mM Tricine-NaOH pH 8.0, 50 mM NaCl and 0.05% β-DDM.
4. Centrifuge the sample in a benchtop centrifuge at 18,407 x g at 4 °C for 5 min to ensure all the material remains in solution.

7. Measuring P700 content of PSI

NOTE: This method can be used to quickly assay PSI.

1. Dilute the samples to an OD₆₈₀ of 1-1.5 and incubate in the dark with 10 mM ascorbic acid for 1 h to reduce P700 completely.
2. Measure the absorption spectrum of the sample using a UV-Vis spectrometer. Measure absorbance from only 650 nm to 750 nm with a data interval of 0.50 nm and a scan rate of 60 nm/min for assaying P700 content.
3. Expose the sample to bright light (350 µmols photons/m²/s) and measure the spectrum again during exposure.
4. Subtract the dark spectrum from the light spectrum to observe P700 bleaching at around 702 nm in plants.
5. Determine the P700 content using the absorption at 700 nm and an extinction coefficient of 64/mM/cm as described in Hiyama and Ke^32,33,34.
6. Measure the total chlorophyll amount (chlorophyll A and chlorophyll B) using the method described in Porra et al.³¹. Use these concentrations to determine the chlorophyll to P700 ratio.

Results

This protocol is used to isolate and characterize active PSI-LHCI from plant tissues over three days. PSI-LHCI is purified by first isolating plant thylakoid membranes which are then solubilized with β-DDM. Typical yields from the membrane preparation stage are 200 mg of chlorophyll from 500 g of leaves. This can vary based on the initial material used.

Days two and three of the experiment use anion exchange chromatography and sucrose gradient centrifugation to separate the different prot...

Discussion

Using this protocol, the PSI-LHCI complex from plant tissues can be purified in its active state. Spinach leaves were used here, but these methods can be applied to preparations from various plants^23,40. In all cases, care must be taken while performing this protocol to protect the complex from damage. This preparation should be done in the dark or under a green light, on ice with pre-chilled buffers, and all resuspension steps should be performed gently.

Materials

Name	Company	Catalog Number	Comments
15 mL Falcon tube	VWR	62406-200	Used for storing thylakoids
Bio rad Econo-Column 1.5 X 30 cm	biorad	7374153
Cheesecloth grade 50, 100% cotton	Arkwright LLC	B07D1FZZMB	From Amazon
Glass rods	Millipore Sigma	BR135825	Any similar rod will suffice
Low profile 64 oz vitamix blender	Vitamix
NaCl	Sigma-Aldrich	S7653
Open top polyallomer centrifugation tubes	Seton Scientific	5030
Optima XE Ultracentrifuge	beckman coulter	A94471
Polyethylene glycol 6,000	Hampton Research	HR2-533
Potter-Elvehjen Tissue Grinder, 30 ml.	WHEATON	358049
Sucrose	Sigma-Aldrich	S7903
SW 40 Ti	beckman coulter	331301
TOYOPEARL DEAE-650C	Tosoh Bioscience	7988
Tricine	Sigma-Aldrich	T0377
β-DDM	Glycon - Biochemicals GmbH	D97002	Stored as 10% stocks at -20 °C