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 th....

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 wi.......

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