Svetla Stoylova

Electron Crystallography of Human Blood Coagulation Factor VIII Bound to Phospholipid Monolayers

Coagulation factor VIII binds to negatively charged platelets prior to assembly with the serine protease, factor IXa, to form the factor X-activating enzyme (FX-ase) complex. The macromolecular organization of membrane-bound factor VIII has been studied by electron crystallography for the first time. For this purpose two-dimensional 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, γ = 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.

Projection structure of photosystem II in vivo determined by Cryo-electron crystallography

Abstract Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4nm, b = 23.1nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.

Structural determination of lipid-bound human blood coagulation factor IX

Human coagulation factor IX (FIX) is a serine protease which binds to a negatively charged phospholipid surface in the presence of Ca ions (Ca2+). FIX two-dimensional (2-D) crystals were obtained by the lipid layer crystallisation technique under near physiological conditions. The 2-D projection map of the protein was calculated to a resolution of 3 nm using electron crystallographic analysis. The structural organisation of membrane-bound FIX is discussed and compared with the known X-ray crystallographic data.

Surfactosomes: a novel approach to the reconstitution and 2-D crystallisation of membrane proteins

The formation of vesicle‐like structures (termed surfactosomes) and lamellar sheets from solutions containing ammonium perfluoroocanoate (APFO) is illustrated using conventional and cryo‐transmission electron microscopy. It is shown how this detergent can be used for the solubilisation, reconstitution, and 2‐D crystallisation of membrane proteins as demonstrated for the major protein of the membrane sector of the V‐type H+‐ATPase (16‐kDa protein). Electron microscopical analysis of 2‐D crystals of the 16‐kDa protein (a = b = 13.0 ± 0.2 nm with γ = 90° and p4 projection symmetry) revealed a unit cell comprising four dimeric complexes of the 16‐kDa protein the significance of which is discussed.

Photosystem II and the Evolution of the Light-harvesting Antennae of Vascular Plants: A New Concept

Cryo-electron crystallography of grana membranes has led toa new concept with regards to the positioning of photosystem II (PSII) relative to its peripheral light-harvesting complex (LHCII). In projection, the structural data reveals small domains surrounding the central PSII core which are compatible with the size and expected stoichiometry of the LHCII proteins. When viewed in 3D, however, these small domains are shown to occupy a membrane separate from the membrane plane that houses the PSII core region. This observation fits with the known morphology of the grana membrane preparation which consists of paired, tightly appressed membranes. The structural data has been confirmed by separate biochemical experiments where LHCII-enriched and core PSII-enriched membrane fractions have been isolated. A vertical segregation of LHCII and PSII within the grana will be of wide significance: (a) Optimisation of light harvesting capacity (packing one membrane with LHCII, whilst at the same time maintaining efficient diffusion of plastoquinone in an adjacent membrane loosely packed with PSII core complexes). (b) Rapid adaptation to changes in light quality and intensity (via physical separation/appression of membrane pairs). (c) Cooperativity (PSII core complexes can tap into a large LHCII antenna located in an adjacent membrane). (d) New understanding of the evolution of light harvesting in plants (cyanobacteria also move light excitation energy vertically from the phycobilisome to PSII core via linker proteins).

Reconstitution of Membranes Using Non-Bilayer Forming Lipids and Plant LHCII

At the molecular level, the structure of the thylakoid membranes is quite complex: they contain specific mixtures of different lipid molecules and a large number of membrane proteins and their aggregates. Thylakoid membranes also display significant structural flexibility, which is associated with their functional activity and with their ability to respond to changes in the environmental conditions [1, 2]. However, our understanding of the structure and dynamics of these membranes is far from complete. Studies on reconstituted systems of purified proteins and lipids may shed light on the basic problem of the self-assembly and structural flexibility of thylakoid membranes [3].