Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.
(17/1790)
Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex. (+info)
Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.
(18/1790)
Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid. (+info)
Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae.
(19/1790)
The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins. (+info)
Physicochemical characterization and purification of cationic lipoplexes.
(20/1790)
Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing. (+info)
Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution.
(21/1790)
Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane alpha-helices, has facilitated the calculation of a projection map of NhaA at 4.0 A resolution. NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of approximately 38x48 A with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter. (+info)
Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.
(22/1790)
Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction. (+info)
Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation.
(23/1790)
The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is approximately 1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of approximately 7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current fluctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel. (+info)
Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography.
(24/1790)
The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules. (+info)