Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Abstract

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, and phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylcholine, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.T e photosynthetic apparatus of green plants comprises a series of integral protein complexes embedded in the thylakoid membrane. The conversion of light energy into a chemically useful form takes place at two reaction centers, photosystem 1 (PSI)' and photosystem 2 (PS2), which consist of complexes of different integral proteins. In the thylakoids from the mesophyll cells of higher plants, the two reaction centers and associated protein complexes are separately located in the appressed (PS2) and nonappressed (PSI) membrane regions. The appressed membranes are arranged in stacks and are interconnected by the nonappressed membranes, which contain regions of high membrane curvature. Within the thylakoid,

Protein assembly and heat stability in developing thylakoid membranes during greening

“…9 and 38. The association constant of SASL relative to MGDG in thylakoid membranes can be estimated from that of SASL and MGDG observed relative to phosphatidylglycerol (21,39) to be K r ϭ Ϸ6.5. With these estimates the values of n are 1.0, 1.1, 0.6, 1, and 2.7 for 0.5, 6, 12, 24, and 48 h of greening, respectively.…”

“…The immobile component was assigned to protein-solvating interfacial lipids (38,39). Because the outer hyperfine splitting of the components differed by more than 5 G, we could determine the immobile fraction ( f i,SASL ) by applying an intersubtraction technique (21,40) on the 34°C spectra, aided with nonlinear least-squares optimization. The intersubtraction leads to excellent fits between the linear combination of the two components and the experimental spectra (Fig.…”

“…Assuming, for simplicity, membrane proteins of the same size, the number of interfacial lipids per monomer protein (N b ) is related to the number of monomers in the protein assembly (oligomer), because of the exclusion of lipids from protein-protein contact regions. A simple model (9,21) can be used to estimate the relative mean oligomer dimensions of these proteins from f i,SASL (20,41). Assuming circular intramembranous intersection for both lipids and proteins of an nxn oligomer, N b is reduced approximately by 1͞n relative to that of a single monomer (for n Ͼ 1), neglecting lipid-trapping sites unavailable for bulk lipids and the label.…”

“…Motional coupling between large, relatively immobile membrane proteins and mobile fluid lipids results in motionally restricted protein-solvating first-shell lipids (19). This appears as ''immobile'' component in EPR spectra of spin-labeled lipid analogues as demonstrated in numerous membrane systems (20), including thylakoids (21). By quantitating these lipids, we have estimated changes in the size of protein assemblies of major thylakoid membrane protein complexes.…”

Differential Detergent Stability of the Major Light-Harvesting Complex II in Thylakoids Isolated from Monocotyledonous and Dicotyledonous Plants

“…Electron spin resonance studies of Li et al (20) have reported that a unique feature of thylakoid membranes is the specificity of interaction of thylakoid PG for thylakoid proteins. PG has been found to be localized primarily in the outer layer of the thylakoid bilayer (22).…”