A Spectroscopic and Cryo-Transmission Electron Microscopy Study

The aggregation behaviour of the cationic pinacyanol chloride in aqueous solution is investigated using absorption and linear dichroism spectroscopies, optical microscopy and cryogenic transmission electron microscopy (cryo-TEM). The investigations are focused on solutions in a concentration range from 50 μM up to 1 mM. At a concentration of 0.7 mM H-aggregates are detected that are characterized by a broad absorption band centred at [similar]511 nm. The aggregates possess a tubular architecture with a single-layer wall thickness of [similar]2.5 nm and an outer diameter of [similar]6.5 nm. Linear dichroism spectroscopy indicates that the molecules are packed with their long axis parallel to the tube axis. These H-aggregates are not stable, but transform into J-aggregates on the time scale of weeks. The kinetics of J-aggregation depends on the dye concentration and the route of sample preparation, but can also be enhanced by shear stress. J-aggregates possess a split absorption spectrum composed of two longitudinally polarized J-bands and one H-band that is polarized perpendicular to the aggregate axis. The J-aggregates are [similar]9 nm wide and several micrometer long fibrils consisting of stacked pairs of ribbons with a dumbbell-shaped density cross-section. Upon aging these ribbons laterally stack face-to-face to form tape-like aggregates

Stereochemistry‐Controlled Supramolecular Architectures of New Tetrahydroxy‐Functionalised Amphiphilic Carbocyanine Dyes

The syntheses of novel amphiphilic 5,5′,6,6‐tetrachlorobenzimidacarbocyanine (TBC) dye derivatives with aminopropanediol head groups, which only differ in stereochemistry (chiral enantiomers, meso form and conformer), are reported. For the achiral meso form, a new synthetic route towards asymmetric cyanine dyes was established. All compounds form J aggregates in water, the optical properties of which were characterised by means of spectroscopic methods. The supramolecular structure of the aggregates is investigated by means of cryo‐transmission electron microscopy, cryo‐electron tomography and AFM, revealing extended sheet‐like aggregates for chiral enantiomers and nanotubes for the mesomer, respectively, whereas the conformer forms predominately needle‐like crystals. The experiments demonstrate that the aggregation behaviour of compounds can be controlled solely by head group stereochemistry, which in the case of enantiomers enables the formation of extended hydrogen‐bond chains by the hydroxyl functionalities. In case of the achiral meso form, however, such chains turned out to be sterically excluded

Towards engineering of self-assembled nanostructures using non-ionic dendritic amphiphiles

Engineering nanostructures of defined size and morphology is a great challenge in the field of self-assembly. Herein we report on the formation of supramolecular nanostructures of defined morphologies with subtle structural changes for a new series of dendritic amphiphiles. Subsequently, we studied their application as nanocarriers for guest molecules

kirjoittanut Hans von Berlepsch ; suomentanut ja suomalaisiin oloihin sovitellut A. J. Mela

Ilmestynyt aikaisemmin saksan, englannin, ranskan, italian ja ruotsin kielillä. Varustettu 8 väritaululla ja 17 tekstikuvall

Charakterisierung der vektoriellen Acylierung als Mechanismus des Fettsäuretransports in Synechocystis sp. PCC 6803

Transfer of fatty acids across biological membranes is a largely uncharacterized process. In unicellular organisms, like Escherichia coli or Saccharomyces cerevisiae vectorial acylation by acyl-activating enzymes is discussed as mechanism for fatty acid transport. In the present work, studies on the only acyl-acyl-carrier protein synthetase SynAas of Synechocystis sp. PCC 6803 with regard to fatty acid transport were performed. Cells deficient in SynAas were highly resistant to externally provided α-linolenic acid. Upon treatment with external α-linolenic acid the wild type showed continuous accumulation of α-linolenic acid in cellular lipids and as free fatty acid in the cell. Long-term incubation with α-linolenic acid for 10 hours led to structural changes in the integrity of thylakoid membranes and a dramatic increase in electron transport rate (ETR) in wild type cells. After 24 hours of incubation the accumulation resulted in bleaching and dying of the wild type cells, whereas the loss of function mutant ∆synaas was completely unaffected by this treatment. Furthermore, Saccharomyces cerevisiae wild type cells displayed sensitivity against α-linolenic acid, whereas the “loss of function” mutant in the fatty acid importer FAT1p showed resistance upon α-linolenic acid incubation. Heterologous expression of SynAas in yeast wild type and ∆fat1 mutant cells resulted in an increased sensitivity against exogenous α-linolenic acid, which implies elevated levels of uptake of toxic α-linolenic acid through SynAas. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate [14C]-α-linolenic acid retrieval from preloaded liposome membranes. Taken together, the data show that the acyl-activating enzyme SynAas is necessary and sufficient to mediate transfer of fatty acids across a biological membrane. These facts were used to derive a model for α-linolenic acid uptake and metabolism in Synechocystis. In addition, the ABC- Transporter SynAbc could be identified as putative fatty acid exporter. The SynAbc “loss-of-function” mutant ∆synabc exhibited increased sensitivity upon α-linolenic acid treatment and massive accumulation of exogenously applied fatty acid, compared to wild type. The ∆synaas mutant secreted free fatty acids into the media. A simultaneous defect in the SynAbc gene completely prevented accumulation of fatty acids in the media, which gives rise to the assumption that SynAbc facilitates fatty acid export in Synechocystis

Vibronic origin of long-lived coherence in an artificial molecular light harvester

Natural and artificial light harvesting processes have recently gained new interest. Signatures of long lasting coherence in spectroscopic signals of biological systems have been repeatedly observed, albeit their origin is a matter of ongoing debate, as it is unclear how the loss of coherence due to interaction with the noisy environments in such systems is averted. Here we report experimental and theoretical verification of coherent exciton-vibrational (vibronic) coupling as the origin of long-lasting coherence in an artificial light harvester, a molecular J-aggregate. In this macroscopically aligned tubular system, polarization controlled 2D spectroscopy delivers an uncongested and specific optical response as an ideal foundation for an in-depth theoretical description. We derive analytical expressions that show under which general conditions vibronic coupling leads to prolonged excited-state coherence

Inhibition of peptide aggregation by means of enzymatic phosphorylation

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP- dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β -sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated

Cyanine Dye Coupling Mediates Self-assembly of a pH Sensitive Peptide into Novel 3D Architectures

Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two-dimensional membrane-like assemblies at pH 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three-dimensional organization

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide

International audienceAmyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements