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

doi:10.1006/abio.1997.2204

Analytical Biochemistry

Volume 250, Issue 2, 1 August 1997, Pages 169-175

https://doi.org/10.1006/abio.1997.2204 Get rights and content

Abstract

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.

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Citation Excerpt :
The isolated BBY membranes were resuspended in media containing 20 mM Mes (pH 6.5), 10 mM NaCl, 5 mM MgCl2, 0.33 M sucrose and stored at −20 °C for further use. LHCsII were isolated following the procedure in [20] using the preparation with 0.7% Triton X-100. Chl content was assayed in 80% acetone (v/v) according to Arnon [21].
The sensitivity of the green plants’ photosystem II (PSII) to high temperatures is investigated in PSII enriched membranes and in membranes, from which the oxygen evolving complex is removed. Using steady-state 77 K fluorescence and resonance Raman spectroscopy we analyze the interdependency between the temperature-driven changes in structure and energy distribution in the PSII supercomplex. The results show that the heat treatment induces different reduction of the 77 K fluorescence emission in both types of investigated membranes: (i) an additional considerable decrease of the overall fluorescence emission in Tris-washed membranes as compared to the native membranes; (ii) a transition point at 42 °C_, observed only in native membranes; (iii) a sharp reduction of the PSII core fluorescence in Tris-washed membranes at temperatures higher than 50 °C; (iv) a 3 nm red-shift of F700 band’s maximum in Tris-washed membranes already at 20 °C and its further shift by 1 nm at temperature increase. Both treatments intensified their action by increasing the aggregation and dissociation of the peripheral light harvesting complexes. The oxygen-evolving complex, in addition to its main function to produce O₂, increases the thermal stability of PSII core by strengthening the connection between the core and the peripheral antenna proteins and by keeping their structural integrity.
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The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers—phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ^av were quantified for a range of quencher concentrations. Applying the Stern–Volmer formalism, apparent quenching rate constants k_q were determined from the dependencies on quencher concentration of the ratio τ₀^av/τ^av, where τ₀^av is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k_q′/k_q, i.e., the apparent quenching rate constants determined in aggregates (or membranes), k_q′, and in detergent-solubilised LHCII trimers, k_q, respectively. In LHCII macroaggregates, the resulting values for the domain size were 15–30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12–24 LHCII trimers, corresponding to 500–1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment–protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.

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

Abstract

Biochim. Biophys. Acta

Trends Biochem. Sci.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Nature

Eur. J. Biochem.

J. Mol. Biol.

Eur. J. Biochem.

Curr. Opin. Struct. Biol.

J. Cell Biol.

Physiol. Plant.

Plant Physiol.

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