Isolation of lamellar aggregates of the light-harvesting chlorophyll a/b protein complex of photosystem II with long-range chiral order and structural flexibility.
Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupa et 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, < or = 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 (deltaCD). (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 (deltaCD). (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 deltaCD. (iv) High concentrations (> or = 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 deltaCD.
Simidjiev
I
I
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Science, Szeged.
Barzda
V
V
Mustárdy
L
L
Garab
G
G
D002942:Circular Dichroism; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D045342:Light-Harvesting Protein Complexes; D008563:Membrane Lipids / Q000737:chemistry; D008854:Microscopy, Electron; D017830:Octoxynol; D018532:Pisum sativum; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D045332:Photosystem II Protein Complex
D003902:Detergents; D045342:Light-Harvesting Protein Complexes; D008563:Membrane Lipids; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D017830:Octoxynol