In many biological membranes, the major lipids are ''non-bilayer lipids,'' which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a͞b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.circular dichroism ͉ chloroplast thylakoid membranes ͉ electron microscopy ͉ light-harvesting complex ͉ lipid-protein interactions T he lamellar organization of biological membranes provides a structural matrix for various proteins and controls the permeability of organic molecules, water, and ions; it also prevents nonspecific protein-protein aggregation, whereas it allows protein diffusion and conformational changes in the membrane. However, biomembranes usually contain substantial amounts of non-bilayer lipids, which in purified form assume nonlamellar structures. In fact, in many membranes, e.g., thylakoid membranes of chloroplasts, membranes of Escherichia coli, rhodopsin, and mitochondria, non-bilayer lipids constitute about half or more of the total lipid content. It is well established that the physical and functional properties of these membranes depend to a large extent on protein-lipid interactions (1, 2). There are a few examples showing that lipid polymorphism can be modulated by proteins, and, in some cases, small unilamellar vesicles can be reconstituted from non-bilayer lipids and membrane proteins (e.g., refs. 3 and 4). However, the structural role of large amounts of non-bilayer lipids has remained enigmatic, and the assembly of extended bilayer lamellae from proteins and predominantly non-bilayer lipids is poorly understood (1, 2, 5). In this work, we use a simple system, the two main constituents of pea thylakoid membranes, purified non-bilayer lipids and isolated protein complexes, to demonstrate that the formation of a large, ordered lamellar structure is possible even in the presence of large amounts of lipids.In chloroplast thylakoid membranes of g...

Coagulation factor VIII binds to negatively charged platelets prior to assembly with the serine protease, factor IXa, to form the factor X-activating enzyme (FXase) complex. The macromolecular organization of membrane-bound factor VIII has been studied by electron crystallography for the first time. For this purpose twodimensional crystals of human factor VIII were grown onto phosphatidylserine-containing phospholipid monolayers, under near to physiological conditions (pH and salt concentration). Electron crystallographic analysis revealed that the factor VIII molecules were organized as monomers onto the lipid layer, with unit cell dimensions: a ‫؍‬ 81.5Å, b ‫؍‬ 67.2 Å, ␥ ‫؍‬ 66.5°, P1 symmetry. Based on a homology-derived molecular model of the factor VIII (FVIII) A domains, the FVIII projection structure solved at 15-Å resolution presents the A1, A2, and A3 domain heterotrimer tilted approximately 65°relative to the membrane plane. The A1 domain is projecting on top of the A3, C1, and C2 domains and with the A2 domain protruding partially between A1 and A3. This organization of factor VIII allows the factor IXa protease and epidermal growth factor-like domain binding sites (localized in the A2 and A3 domains, respectively) to be situated at the appropriate position for the binding of factor IXa. The conformation of the lipid-bound FVIII is therefore very close to that for the activated factor VIIIa predicted in the FX-ase complex.Factor VIII (FVIII) 1 is an essential protein in blood coagulation (1). Deficiency in FVIII is responsible for hemophilia A (classic hemophilia), an X-chromosome-linked bleeding disorder (2). During coagulation, FVIII is proteolytically cleaved to an unstable active heterodimeric form (FVIIIa) by trace amounts of thrombin or factor Xa (FXa) (3). FVIIIa functions as a cofactor, responsible for the efficient activation of factor X (FX) by activated factor IX (FIXa) in the presence of negatively charged phospholipids (PL) and Ca 2ϩ ion (4). The assembly of the membrane-bound FX-ase complex (FX/FVIIIa/FIXa) results in the release of FXa, which associates with the cofactor factor Va (FVa) and prothrombin into the membrane-bound prothrombinase complex. The released thrombin further cleaves fibrinogen to insoluble fibrin, which stabilizes the growing clot and arrests bleeding (5).The binding of FVIIIa to FIXa enhances the catalytic reaction by at least 5 orders of magnitude (6). Although the molecular mechanism of this amplifying effect is not yet known, it seems to be strongly related to the formation of the FX-ase complex onto negatively charged membranes: increasing both the collision frequencies by restricting the reactants to a twodimensional surface, and the specificity of the FIXa active site (7,8).Human FVIII is a large plasma glycoprotein synthesized as a single-chain polypeptide with a predicted molecular mass of 264,763 Da, corresponding to the mature 2332 amino acid sequence, determined from translation of the cloned gene. With N-linked carbohydrate chains, the molecular mass ...

Electron crystallography constitutes a powerful new method for determining the structure of biological macromolecules. This method is best adapted to the study of ordered assemblies of macromolecules, and principally to two-dimensional (2-D) crystals of proteins. Obtaining protein 2-D crystals ordered at high resolution constitutes the major limiting step in the application of this approach. Considerable interest has been raised by the development of a rational method of 2-D crystallization based on the specific binding of proteins to planar lipid films. The applicability of this method is quasi-general in the case of soluble proteins. Its basic principles, together with examples taken from work in our group, are presented here.

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, ~, = 107 °. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm x 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked ÔgranaÕ regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.

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