The 3D structure of rat DPPIV/CD26 as obtained by cryo-TEM and single particle analysis

We present the three-dimensional structure of rat DPPIV/CD26, as determined by cryo-TEM and single particle analysis at a resolution of approximately 14A. The reconstruction confirms that the protein exists as a dimer, as predicted earlier. Since there are structural analogies to the serine peptidase POP, docking calculations of the two structures were performed. Although the docking showed a similar spatial organization (catalytic domain, beta-propeller, distal opening, central cavity), the detailed comparison revealed clear discrepancies. The most marked difference is a second (lateral) opening in DPPIV/CD26, which would enable direct access to the catalytic site. We therefore assume that substrate selectivity and binding rate are most probably driven by different mechanisms in DPPIV/CD26 and POP.     

Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.     

Thickness distribution of actin bundles in vitro

Bundles of filamentous actin form the primary building blocks of a broad range of cytoskeletal structures, including filopodia, stereocilia and microvilli. In each case, the cell uses specific associated proteins to tailor the dynamics, dimensions and mechanical properties of the bundles to suit a specific cellular function. While the length distribution of actin bundles was extensively studied, almost nothing is known about the thickness distribution. Here, we use high-resolution cryo-TEM to measure the thickness distribution of actin/fascin bundles, in vitro. We find that the thickness distribution has a prominent peak, with an exponential tail, supporting a scenario of an initial fast formation of a disc-like nucleus of short actin filaments, which only later elongates. The bundle thicknesses at steady state are found to follow the distribution of the initial nuclei indicating that no lateral coalescence occurs. Our results show that the distribution of bundles thicknesses can be controlled by monitoring the initial nucleation process. In vivo, this is done by using specific regulatory proteins complexes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel properties of cholesterol-dioleoylphosphatidylcholine mixtures

We have studied the properties of mixtures of cholesterol with dioleoylphosphatidylcholine (DOPC), and with several other phospholipids, including 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and dioleoleoylphosphatidylserine (DOPS), as a function of cholesterol molar fraction and of temperature. Mixtures of DOPC with a cholesterol molar fraction of 0.4 or greater display polymorphic behavior. This polymorphism includes the formation of structures that give rise to isotropic peaks in ³¹P NMR at cholesterol molar fractions between 0.4 and 0.6, dependent on the thermal history of the sample. Cryo-electron microscopy studies demonstrate the formation of small globular aggregates that would contribute to a narrowing of the ³¹P NMR powder pattern.At molar fraction cholesterol 0.6 and higher and at temperatures above 70 °C, the mixtures with DOPC convert to the hexagonal phase. Lipid polymorphism is accompanied by the phase separation of cholesterol crystals in the anhydrous form and/or the monohydrate form. The crystals that are formed have substantially altered kinetics of hydration and dehydration, compared with both pure cholesterol monohydrate crystals and with crystals formed in the presence of the other phospholipids that do not form the hexagonal phase in the presence of cholesterol. This fact demonstrates that these cholesterol crystals are in intimate contact with the DOPC phospholipid and are not present as morphologically separate structures.     

Unilamellar vesicles obtained by simply mixing dioctadecyldimethylammonium chloride and bromide with water

Optically clear dispersions of dioctadecyldimethylammonium bromide and chloride (DODAX, X = Br⁻, Cl⁻) in water can be obtained by simply mixing the amphiphiles at low concentrations (1 mM) and at a temperature safely above the gel to liquid crystalline phase transition temperature (T_m ≈ 45–48 °C) of DODAX in water. Under these conditions, dynamic light scattering shows that, at room temperature, the dispersions contain two well-defined populations of large vesicles with average hydrodynamic radii (R_H) of 80 and 337 nm for DODAB and of 69 and 247 nm for DODAC. Cryo-transmission electron microscopy (cryo-TEM) micrographs show that DODAX vesicles are unilamellar and polydisperse with apparent radius up to 800 nm. The vesicles are stable for at least 1 month according to the ageing time-dependence of the turbidity and molar absorption coefficient.     

Solubilization of sphingomyelin vesicles by addition of a bile salt

The interactions of the bile salt sodium taurocholate (TC) in 50 mM Trizma-HCl buffer and 150 mM NaCl (pH 9) at 37 degrees C with membranes composed of sphingomyelin (SM) were studied by dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM) and turbidity measurements. Small unilamellar SM vesicles were prepared by extrusion. Below the CMC of TC, taurocholate addition leads to vesicle growth due to incorporation of the taurocholate molecules into the vesicle bilayer. At around half the CMC of the bile salt, the SM vesicles are transformed into SM/TC mixed worm-like micelles, which are visualized by cryo-TEM for the first time. Further increase in the taurocholate concentration leads to the rupture of these structures into small spherical micelles. Interestingly, large non-spherical micelles were also identified for pure taurocholate solutions. Similar threadlike structures have been reported earlier for the bile salt sodium taurodeoxycholate [Rich, A., Blow, D., 1958. Nature 182, 1777; Blow, D.M., Rich, A., 1960. J. Am. Chem. Soc. 82, 3566-3571; Galantini, L., Giglio, E., La Mesa, C., Viorel-Pavel, N., Punzo, F., 2002. Langmuir 18, 2812] and for mixtures of taurocholate and phosphatidylcholate [Ulmius, J., Lindblom, G., Wennerstr?m, H., Johansson, L.B.-A., Fontel, K., S?derman, O., Ardvisson, G., 1982. Biochemistry 21, 1553; Hjelm, R.P., Thiyagarajan, P., Alkan-Onyuksel, H., 1992. J. Phys. Chem. 96, 8653] as determined by various scattering methods.     

Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

Synergistic cosolubilization of omega-3 fatty acid esters and CoQ10 in dilutable microemulsions

Water-dilutable microemulsions were prepared and loaded with two types of omega-3 fatty acid esters (omega-3 ethyl esters, OEE; and omega-3 triacylglycerides, OTG), each separately and together with ubiquinone (CoQ₁₀). The microemulsions showed high and synergistic loading capabilities. The linear fatty acid ester (OEE) solubilization capacity was greater than that of the bulky and robust OTG.The location of the guest molecules within the microemulsions at any dilution point were determined by electrical conductivity, viscosity, DSC, SAXS, cryo-TEM, SD-NMR, and DLS.We found that OEE molecules pack well within the surfactant tails to form reverse micelles that gradually, upon water dilution, invert into bicontinuous phase and finally into O/W droplets. The CoQ₁₀ increases the stabilization and solubilization of the omega-3 fatty acid esters because it functions as a kosmotropic agent in the micellar system. The hydrophobic and bulky OTG molecule strongly interferes with the tail packing and spaces them significantly – mainly in the low and medium range water dilutions. When added to the micellar system, CoQ₁₀ forms some reverse hexagonal mesophases. The inversion into direct micelles is more difficult in comparison to the OEE system and requires additional water dilution. The OTG with or without CoQ₁₀ destabilizes the structures and decreases the solubilization capacity since it acts as a chaotropic agent to the micellar system and as a kosmotropic agent to hexagonal packing. These results explain the differences in the behavior of these molecules with vehicles that solubilize them in aqueous phases.Temperature disorders the bicontinuous structures and reduces the supersaturation of the system containing OEE with CoQ₁₀; as a result CoQ₁₀ crystallization is retarded.     

3D structure of the CD26-ADA complex obtained by cryo-EM and single particle analysis

The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.