Structural Mechanism and Photoprotective Function of Water-soluble Chlorophyll-binding Protein

Daisuke Horigome,Hiroyuki Satoh,Nobue Itoh,Katsuyoshi Mitsunaga,Isao Oonishi,Atsushi Nakagawa,Akira Uchida

doi:10.1074/jbc.m609458200

Daisuke Horigome, Hiroyuki Satoh + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m609458200

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Abstract

A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.

Highlights

Water-soluble chlorophyll-binding proteins (WSCPs),4 which form a complex with chlorophyll (Chl), have been isolated from Amaranthaceae, Chenopodiaceae, and Polygonaceae and from Brassicaceae
This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function
Protein Preparation and Crystallization—WSCP-Chl complex was prepared from the leaves of L. virginicum cultivated in Chiba, Japan, and purification was performed by a combination of ammonium sulfate precipitation, anion exchange chromatography, detergent-free polyacrylamide gel electrophoresis, and gel filtration chromatography as described in (14 –16) with some modifications

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation and Crystallization—WSCP-Chl complex was prepared from the leaves of L. virginicum cultivated in Chiba, Japan, and purification was performed by a combination of ammonium sulfate precipitation, anion exchange chromatography, detergent-free polyacrylamide gel electrophoresis, and gel filtration chromatography as described in (14 –16) with some modifications. The modifications were that the fraction of 40 –90% ammonium sulfate precipitation of the homogenate was separated by DE52 DEAE cellulose chromatography (Whatman Plc, Brentford, UK) in which the elution buffer was 0.2 M sodium/potassium phosphate, pH 7.0, and the final gel filtration chromatography with a Sephacryl S-200 HR (GE Healthcare) was performed with 0.1 M sodium/potassium phosphate pH 7.0. Crystals of WSCP-Chl complex were grown by means of vapor diffusion by mixing 2 ␮l of a protein solution containing 0.1 M sodium/potassium phosphate, pH 6.0, with 2 ␮l of a reservoir solution containing 5% sucrose, 3.2 M ammonium sulfate, and 0.1 M sodium/potassium phosphate, pH 6.0, at 20 °C. The root mean square deviations of C␣ atoms were calculated with the program LSQKAB in CCP4 (18), the volume of the Chlbinding cavity with the program VOIDOO (24), and the wateraccessible surface area and buried molecular surface area with the program SurfRace (25)

RESULTS

Ramachandran plot

DISCUSSION