Formation of crystalline arrays of chlorophyll a/b - light-harvesting protein by membrane reconstitution

Abstract

The structure of the major protein constituent of photosynthetic membranes in higher plants, the chlorophyll a/b-light harvesting complex (LHC), was studied by x-ray diffraction and electron microscopy. The LHC was purified from Triton X-100 solubilized thylakoid membranes of the pea, and contained 6 mol of chlorophylls a and b per mole of a polypeptide of 27,000 molecular weight. X-ray diffraction showed that in the presence of 10 mM MgCl2, purified LHC forms planar aggregates that stack with a period of 51 A. Within each layer, LHC molecules pack with a center-to-center distance of 85 A but without long-range order. However, when LHC is incorporated into single-walled vesicles of plant lecithin, the addition of NaCl above 10 mM, or MgCl2 above 2 mM, led to the formation of plaques of hexagonal lattices, with a lattice constant of 125 A. The large domain size and high degree of order in the plane of the membrane are evident from the sharp lattice lines observed to 7 A resolution on the equator of the x-ray pattern. Freeze-fracture electron micrographs demonstrated an aligned stacking of the lattices in adjacent membranes, resulting in crystallinity in the third dimension over short distances. Micrographs of negatively stained membranes revealed a hexagonal lattice of the same lattice constant, formed by surface-exposed parts of the LHC molecules which are probably responsible for the ordered stacking of lattices. In both the LHC aggregates and in the reconstituted membrane lattices the diffracted x-ray intensities at 10-A spacing on the equator indicate that the LHC molecule contains paralled alpha-helices or beta-sheets that are oriented perpendicular to the planar arrays.

Chloroplast Membrane Protein Phosphorylation

“…It has been estimated that one LHC-I1 structural unit (see Fig. 1D) consists of 4-7 individual polypeptides of 25-29 kdaltons, each of which binds 6-13 Chl a + b in the native complex [21,44,49]. A surface-exposed segment of the LHC-I1 (amino-acid sequence shown in Fig.…”

Light-harvesting chlorophyll a / b -protein: Three-dimensional structure of a reconstituted membrane lattice in negative stain

“…The purified protein has been incorporated into phospholipid vesicles and induced to crystallize in the plane of the lipid bilayer (13)(14)(15)(16). Using x-ray diffraction and electron microscopy, Li and Hollingshead (16) showed that LHC lattices in the reconstituted membranes are hexagonal, with a lattice constant of 125 A and a thickness of 70 A.…”

“…In this study, the LHC lattices have been isolated from the reconstituted membranes (16) by eliminating the contiguous pure lipid phase. The three-dimensional structure of the lattice in negative stain has been determined by image reconstruction from electron micrographs (28,30).…”

Nuclear, Virescent Mutants of Zea mays L. with High Levels of Chlorophyll (a/b) Light-Harvesting Complex during Thylakoid Assembly

“…Poor lipid synthesis might generate the pleiotropic phenotype of v*-424. LHC is one of few membrane proteins that self-assembles into sheets in the absence of polar glycerolipid (19) and thus might be less affected than other thylakoid complexes. We analyzed the lipid content of developing mutant thylakoids in order to ascertain whether there was any reduction of glycerolipid content that accompanied the greatly reduced levels of most thylakoid polypeptides.…”