Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

• Published in: Analytical biochemistry Vol. 250; no. 2; pp. 169 - 175
• Main Authors: Simidjiev, Ilian, Barzda, Virginijus, Mustárdy, László, Garab, Győző
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-08-1997
• Subjects: Circular Dichroism; Detergents; Electrophoresis, Polyacrylamide Gel; Light-Harvesting Protein Complexes; Membrane Lipids - chemistry; Microscopy, Electron; Octoxynol; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - isolation & purification; Photosynthetic Reaction Center Complex Proteins - ultrastructure; Photosystem II Protein Complex; Pisum sativum
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1997.2204

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupaet al.(1987,Plant Physiol.84, 19–24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, ≤0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (ΔCD). (ii) At the optimal concentration, around 0.7 ± 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0–1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (ΔCD). (iii) At detergent concentration above the optimum, around 0.8–1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor ΔCD. (iv) High concentrations (≥1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no ΔCD.