Christoph Boettcher

Electron radiation damage to protein crystals of bacteriorhodopsin at different temperatures

Abstract A series of diffraction patterns from two-dimensional protein crystals of bacteriorhodopsin (purple membrane) at different temperatures (294 K, 98 K and 4 K) were recorded as the diffraction spots faded due to radiation damage. The patterns were then computationally evaluated in order to obtain a quantitative measurement of the structural preservation while irradiating the specimen. To provide statistically significant results, diffraction spots corresponding to spacings of 3A(1200 spots) and 7A(600 spots) were measured. A substantial increase of the lifetime of high resolution spots was found using liquid nitrogen as a coolant, whereas further structural preservation at liquid helium temperature was significant but smaller. It appears likely therefore that high resolution images are accessible even at liquid nitrogen temperature. Mechanical stability and the absence of thermal specimen drift are certainly of equivalent importance for successful high resolution imaging.

Stacked bilayer helices: a new structural organization of amphiphilic molecules

Intriguing helical fibres can be created by self-assembly of simple chiral amphiphilic molecules. We study the parameters governing this spontaneous self-organization by three-dimensional (3D) electron microscopy of the helical fibres embedded in a vitreous ice-matrix. Different stable helices are generated reproducibly using specific combinations of the control parameters in our system. All fibres with diameters less than 25 nm consist of a narrow stack of compartmented bilayers twisted into a left-handed helix. Our novel helical 3D reconstruction procedures in combination with specialized cryomicroscopical specimen preparation, can rapidly elucidate the structure of such helical assemblies. This approach may complement or even replace existing diffraction-based methodologies.

A Novel Green Template for the Synthesis of Mesoporous Silica

Mesoporous pure silicas and functionalized silica with a narrow pore size distribution centered at 3.8 nm were prepared by a novel template, amphiphilic dendritic polyglycerol. The resulting silica materials were characterized by electron microscopy; nitrogen adsorption; (1)H, (13)C, and (29)Si solid-state cross-polarization magic-angle spinning NMR spectroscopy. It was shown that the template could be completely removed from the pure and functionalized silica in an environmentally friendly way by means of a simple water extraction procedure. Furthermore, it was shown that these materials could be easily functionalized, for example, by employing aminopropyl groups. Thus, a new environmentally friendly pathway to this fascinating class of silica material has been opened.

Flexible loops of thread-like micelles are formed upon interaction of l-α-dimyristoyl-phosphatidylcholine with the biosurfactant surfactin as revealed by cryo-electron tomography

Vesicles of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) are known to disintegrate upon treatment with surfactin, a lipoheptapeptide biosurfactant from Bacillus subtilis OKB 105, as was observed by static light scattering (SLS) and cryo-transmission electron microscopy (cryo-TEM) recently. The lysis of DMPC bilayers occurs strongly dependent on the surfactin concentration according to a three-stage model. Unilamellar DMPC vesicles are disrupted to form sheet-like lamellar intermediates at a moderate surfactant concentration, but undergo a transition towards smaller particles of unknown structure at a higher surfactant concentration according to earlier neutron scattering experiments. Here we present direct structural evidence from cryo-electron tomography data that thread-like micelles with a uniform diameter of 6.5 nm are organized into loops of different sizes at a surfactin concentration of > 15 mol%.

Human Serum Albumin Nanotubes with Esterase Activity

A nanocylindrical wall structure was obtained by layer-by-layer (LbL) assembly of poly-L-arginine (PLA) and human serum albumin (HSA) and characterized by scanning electron microscopy (SEM), scanning force microscopy (SFM), and cryogenic transmission electron microscopy (cryo-TEM). SEM and SFM measurements of a lyophilized powder of (PLA/HSA)(3) nanotubes yielded images of round, chimney-like architectures with approximately 100 nm wall thickness. Cryo-TEM images of the hydrated sample revealed that the tube walls are composed of densely packed HSA molecules. Moreover, when small-angle X-ray scattering was used to characterize the individual PLA and HSA components in aqueous solutions, maximum diameters of approximately 28 nm and 8 nm were obtained, respectively. These values indicate the minimum thickness of wall layers consisting of PLA and HSA. It can also be concluded from SEM as well as from cryo-TEM images that the protein cylinders are considerably swollen in the presence of water. Furthermore, HSA retains esterase activity if assembled in nanotubes, as indicated by measurements of para-nitrophenyl acetate hydrolysis under semi-physiological conditions (pH 7.4, 22 °C). The enzyme activity parameters (Michaelis constant, K(m), and catalytic constant, k(cat)) were comparable to those of free HSA.

Physical properties and packing states of molecular assemblies of synthetic glycolipids in aqueous dispersions

Amidic glycolipids, 1,5-bis-O-alkyl-N-maltooligonoyl-L-glutamate (1), having various lengths of two hydrocarbon chains (carbon number, m: 14, 16, 18) and maltooligotose with (glucose unit, n: 3, 5, 7) and a N-glycosidic lipid, 1,5-bis-O-octadecyl-N-maltopentaonosyl-L-glutamate (2) have been synthesized. The assembling structures were analyzed by microscopic observation, such as negatively stained TEM, cryo-TEM, and AFM. The glycolipid 1a (m,n: 14,5) showed a fiber-like structure in all the observed temperatures, while 1b (16,5) showed a fiber-like structure when the hydrating temperature was above the gel-to-liquid crystalline phase transition temperature (Tc; 45°C) and a large disk-like structure when incubated below the Tc. The glycolipid 1c (18,5) took a large disk-like structure after hydration of the powder above the Tc. The glycolipids 1d (18,3) and 1e (18,7) showed a mixture of large disks and large vesicles and a mixture of small disks and micelles, respectively. The N-glycosidic lipid, 2, with no amide linkage made a vesicular structure only. The preparation procedure using high shear stress, such as extrusion and sonication, converted the large disk of 1c to smaller assemblies, such as small disk-, cone-, and granule-like assemblies, depending on the preparation conditions. The glycolipid molecules in the planer part of the disk were packed so tightly that molecular mobility was very low even above the Tc (58°C), and the reactivity of the saccharide chain against Concanavalin A was also very low, indicating that the high reactivity probably comes from the loose packing of saccharide chains around the edge part of the assemblies.

Chemo-Enzymatic Synthesis of Perfluoroalkyl-Functionalized Dendronized Polymers as Cyto-Compatible Nanocarriers for Drug Delivery Applications

Among amphiphilic polymers with diverse skeletons, fluorinated architectures have attracted significant attention due to their unique property of segregation and self-assembly into discrete supramolecular entities. Herein, we have synthesized amphiphilic copolymers by grafting hydrophobic alkyl/perfluoroalkyl chains and hydrophilic polyglycerol [G2.0] dendrons onto a co-polymer scaffold, which itself was prepared by enzymatic polymerization of poly[ethylene glycol bis(carboxymethyl) ether]diethylester and 2-azidopropan-1,3-diol. The resulting fluorinated polymers and their alkyl chain analogs were then compared in terms of their supramolecular aggregation behavior, solubilization capacity, transport potential, and release profile using curcumin and dexamethasone drugs. The study of the release profile of encapsulated curcumin incubated with/without a hydrolase enzyme Candida antarctica lipase (CAL-B) suggested that the drug is better stabilized in perfluoroalkyl chain grafted polymeric nanostructures in the absence of enzyme for up to 12 days as compared to its alkyl chain analogs. Although both the fluorinated as well as non-fluorinated systems showed up to 90% release of curcumin in 12 days when incubated with lipase, a comparatively faster release was observed in the fluorinated polymers. Cell viability of HeLa cells up to 95% in aqueous solution of fluorinated polymers (100 μg/mL) demonstrated their excellent cyto-compatibility.

Assignment of two ultrastructures formed by a mixture of hexonamides using autoradiography and electron microscopy

The combined application of autoradiography and electron microscopy allowed the assignment of molecular components to individual micellar fibres in a mixed gel. Resolution was of the order of 0.1 μm. As a result, it was shown that bimolecular sheets of N‐dodecyl‐l‐mannonamide (= l‐Man‐12) completely separated from helical rods consisting of tritiated N‐octyl‐d‐gluconamide (= d‐Glu‐8). The method should be useful for the analysis of several other synthetic supramolecular systems.

Vis- and CD-spectroscopic studies of the interaction of amphiphiles with optically active J-aggregates

J-aggregates are useful model systems for investigations about photosynthesis and photoactive bacteria [1]. The achiral monomeric benzimidocarbocyanine C8O3 (Fig. 1) forms optically active J-aggregates in aqueous solution [2]. Their chiral supramolecular structure can be influenced by cationic and anionic amphiphiles which is shown by vis-, circular dichroism (CD)-spectroscopy and cryo-electron microscopy (cryo-EM).