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.

NFGAIL Amyloid Oligomers: The Onset of Beta-Sheet Formation and the Mechanism for Fibril Formation.

The hexapeptide NFGAIL is a highly amyloidogenic peptide, derived from the human islet amyloid polypeptide (hIAPP). Recent investigations indicate that presumably soluble hIAPP oligomers are one of the cytotoxic species in type II diabetes. Here we use thioflavin T staining, transmission electron microscopy, as well as ion mobility-mass spectrometry coupled to infrared (IR) spectroscopy to study the amyloid formation mechanism and the quaternary and secondary structure of soluble NFGAIL oligomers. Our data reveal that at neutral pH NFGAIL follows a nucleation dependent mechanism to form amyloid fibrils. During the lag phase, highly polydisperse, polymorph, and compact oligomers (oligomer number n = 2-13) as well as extended intermediates (n = 4-11) are present. IR secondary structural analysis reveals that compact conformations adopt turn-like structures, whereas extended oligomers exhibit a significant amount of ß-sheet content. This agrees well with previous molecular dynamic simulations and provides direct experimental evidence that unordered off-pathway NFGAIL aggregates up to the size of at least the 13-mer as well as partially folded ß-sheet containing oligomers are coexisting.

Introducing Chirality into Nonionic Dendritic Amphiphiles and Studying Their Supramolecular Assembly.

Chiral head groups have been introduced into water-soluble hydroxyl-terminated nonionic amphiphiles and the impact of the head group stereochemistry on the supramolecular ultrastructures has been studied. Enantiomeric isomers were compared with the achiral meso form and the racemic mixture by means of cryogenic transmission electron microscopy and circular dichroism spectroscopy. Structurally, all amphiphiles are composed of the first-generation hydrophilic polyglycerol head group coupled to a single hydrophobic hexadecyl chain through an amide linkage and diaromatic spacer. The enantiomers aggregate to form twisted ribbons with uniform handedness, whereas the meso stereoisomer and racemic mixture produce elongated assemblies, namely, tubules and platelets, but without a chiral ultrastructure. Simulations on the molecular packing geometries of the stereoisomers indicate different preferential assembly routes that explain the individual supramolecular aggregation behavior.

Vibronic and vibrational coherences in two-dimensional electronic spectra of supramolecular J-aggregates.

In J-aggregates of cyanine dyes, closely packed molecules form mesoscopic tubes with nanometer-diameter and micrometer-length. Their efficient energy transfer pathways make them suitable candidates for artificial light harvesting systems. This great potential calls for an in-depth spectroscopic analysis of the underlying energy deactivation network and coherence dynamics. We use two-dimensional electronic spectroscopy with sub-10 fs laser pulses in combination with two-dimensional decay-associated spectra analysis to describe the population flow within the aggregate. Based on the analysis of Fourier-transform amplitude maps, we distinguish between vibrational or vibronic coherence dynamics as the origin of pronounced oscillations in our two-dimensional electronic spectra.

Biosupramolecular nanowires from chlorophyll dyes with exceptional charge-transport properties.

Conductive tubes: Self-assembled nanotubes of a bacteriochlorophyll derivative are reminiscent of natural chlorosomal light-harvesting assemblies. After deposition on a substrate that consists of a non-conductive silicon oxide surface (see picture, brown) and contacting the chlorin nanowires to a conductive polymer (yellow), they show exceptional charge-transport properties.

Inhibition of amyloid aggregation by formation of helical assemblies.

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-ß-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a ß-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a ß-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.

Structure analysis of an amyloid-forming model peptide by a systematic glycine and proline scan.

The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high ß-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the ß-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.

Aggregation of Amphiphilic Carbocyanines: Fluorination Favors Cylindrical Micelles over Bilayered Tubes.

The synthesis of a new amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine dye derivative with -(CH2)2-(CF2)5-CF3 chains attached to the nitrogen atoms in the 1,1'-position, CF8O3, is reported. Depending on the dye concentration and the addition of MeOH, CF8O3 forms J- and H-aggregates in aqueous solutions. The aggregation behavior was investigated using steady-state absorption, linear dichroism, and fluorescence spectroscopy, as well as by cryogenic transmission electron microscopy (cryo-TEM). The J-band of the MeOH-free solution is monomer-like, rather broad, and less red-shifted with respect to the monomer absorption, indicating weak excitonic coupling and disorder effects. Cryo-TEM reveals a diversity of supramolecular structures, wherein linear and branched cylindrical micelles dominate. It is concluded that the high stiffness of fluoroalkyl chains does not allow the chains to splay and completely fill up the hydrophobic gap between opposing chromophores. This destabilizes the bilayers and favors the micellar structure motifs instead. The aggregates appearing at 30% MeOH show a split absorption spectrum consisting of a broad blue-shifted H-band and an accompanying sharp red-shifted J-band with perpendicular polarizations. These HJ-type aggregates are also composed of micellar fibers, but these bundle into rope-like strands. For 10% MeOH, a narrow bilayered tube is the dominating morphology. The observed MeOH dependence of aggregation reveals a clear cosolvent effect.

Peptide adsorption to cyanine dye aggregates revealed by cryo-transmission electron microscopy.

The binding interaction between aggregates of the 5-chloro-2-[[5-chloro-3-(3-sulfopropyl)-3H-benzothiazol-2-ylidene]methyl]-3-(3-sulfopropyl)benzothiazolium hydroxide inner salt ammonium salt (CD-1) and alpha-helix, as well as beta-sheet forming de novo designed peptides, was investigated by absorption spectroscopy, circular dichroism spectroscopy, and cryogenic transmission electron microscopy. Both pure dye and pure peptides self-assembled into well-defined supramolecular assemblies in acetate buffer at pH = 4. The dye formed sheetlike and tubular H- and J-aggregates and the peptides alpha-helical coiled-coil assemblies or beta-sheet rich fibrils. After mixing dye and peptide solutions, tubular aggregates with an unusual ultrastructure were found, most likely due to the decoration of dye tubes with monolayers of peptide assemblies based on the strong electrostatic attraction between the oppositely charged species. There was neither indication of a transfer of chirality from the peptides to the dye aggregates nor the opposite effect of a structural transfer from dye aggregates onto the peptides secondary structure.

How metal ions affect amyloid formation: Cu2+- and Zn2+-sensitive peptides.

The common feature of proteins involved in many neurodegenerative diseases is their ability to adopt at least two different stable conformations. The conformational transition that shifts the equilibrium from the functional, mostly partially alpha-helical structure, to the beta-sheet rich amyloid can be triggered by numerous factors, such as mutations in the primary structure or changes in the environment. We present a set of model peptides that, without changes in their primary structure, react in a predictable fashion in the presence of transition metal ions by adopting different conformations and aggregate morphologies. These de novo designed peptides strictly follow the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. Furthermore, domains that favor beta-sheet formation have been incorporated to make the system prone to amyloid formation. As a third feature, histidine residues create sensitivity towards the presence of transition metal ions. CD spectroscopy, ThT fluorescence experiments, and transmission electron microscopy were used to characterize peptide conformation and aggregate morphology in the presence of Cu2+ and Zn2+. Furthermore, the binding geometry within peptide-Cu2+ complexes was characterized by electron paramagnetic resonance spectroscopy.

Intramolecular charge interactions as a tool to control the coiled-coil-to-amyloid transformation.

Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insoluble beta-sheet rich fibrils. Molecular-level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the beta-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH-controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted alpha-helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid-like fibrils. Peptide B features a glutamate-based switch functionality and exhibits opposite pH-dependent folding behavior. In this case, alpha-helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.

Modification of the nanoscale structure of the J-aggregate of a sulfonate-substituted amphiphilic carbocyanine dye through incorporation of surface-active additives.

The amphiphilic dye 3,3'-bis(2-sulfopropyl)-5,5',6,6'-tetrachloro-1,1'-dioctylbenzimidacarbocyanine (C8S3) self-aggregates in aqueous solution to form tubular J-aggregates with a diameter of 17.0 +/- 0.5 nm, a wall thickness of approximately 4 nm, and a length exceeding several hundred nanometers. The absorption spectrum shows the typical features expected for tubular J-aggregates with several sharp and red-shifted absorption bands. Morphological investigations using cryo-transmission electron microscopy (cryo-TEM) and spectroscopic investigations reveal a high stability of the tubular morphology but a tendency of the aggregates to assemble into ropelike bundles after several weeks of storage. It is found that aggregation in solutions containing additives such as alcohols or surfactants results in the formation of new types of aggregates. A second type of tubular aggregate with a diameter of 13.0 +/- 0.5 nm is observed when the solutions contain more than 10 wt % MeOH. On the time scale of days these tubular aggregates transform into ribbonlike structures characterized by a new absorption spectrum, and they convert after several weeks into giant tubes with diameters of up to 500 nm.

Investigation of a dual set of driving forces (hydrophobic + electrostatic) for the two-step fabrication of defined block copolymer micelles.

The aqueous complexation of an amphiphilic block copolymer AB containing a hydrophobic segment A and a polyanionic segment B, with a double hydrophilic block copolymer CD containing a polycationic block C and a non-ionic block D was studied. Defined A(BC)D aggregates ( spherical micelles and vesicles) were obtained by this novel sequential pathway superposing both hydrophobic and electrostatic forces. This proof of concept indicates that this straightforward strategy could be further applied for preparing compartmented polymer-based nanostructures.

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.

Structural changes of poly(butadiene)-poly(ethyleneoxide) diblock-copolymer micelles induced by a cationic surfactant: scattering and cryogenic transmission electron microscopy studies.

Micelles of the diblock copolymer poly(butadiene)-poly(ethyleneoxide) (B40-b-EO62) and mixed micelles of this polymer with the cationic surfactant dodecyltrimethylammonium bromide (C12TAB) were investigated using static and dynamic light scattering and small-angle neutron scattering. It is shown that the surfactant induces a major structural change from large mainly rodlike aggregates to smaller spherical mixed micelles. The rodlike assemblies found in the absence of surfactant have a contour length L of ca. 500 nm and a diameter d approximately 30 nm. The spherical mixed micelles obtained upon addition of C12TAB possess a hydrodynamic radius of 15 nm and still contain several polymer molecules. The results of the scattering experiments are consistent with observations of the aggregates by cryogenic transmission electron microscopy.

Specific in situ discrimination of amyloid fibrils versus α-helical fibres by the fluorophore NIAD-4.

A wide range of human pathologies, including neurodegenerative diseases and other forms of amyloidosis, are associated with the formation of insoluble fibrillar protein aggregates known as amyloids. To gain insights into this process analytical methods are needed, which give quantitative data on the molecular events that are taking place. The dye Thioflavin T (ThT) is widely used for the spectroscopic determination of amyloid fibril formation. Different binding affinities to amyloids at neutral and acidic pH and the frequently observed poor binding at acidic pH are problematic in the use of the cationic ThT. The uncharged fluorescence probe [[5'-(4-hydroxyphenyl)[2,2'-bithiophen]-5-yl]methylene]-propanedinitrile (NIAD-4) has been recently designed by Swager and coworkers, in order to eliminate some of the limitations of ThT. Here we have used this novel dye for in vitro monitoring of the amyloid formation processes of de novo designed model peptides. Amyloid structures were successfully detected by NIAD-4 at neutral as well as acidic pH and no significant fluorescence was detectable in the presence of α-helical fibres. Thus, NIAD-4 proved to be a valuable alternative to ThT for spectroscopic studies on amyloid structures over a broad pH range.

Two-compartment micellar assemblies obtained via aqueous self-organization of synthetic polymer building blocks.

We synthesized a symmetric linear ABCBA pentablock copolymer consisting of poly(ethylene oxide), poly(gamma-benzyl l-glutamate), and a poly(perfluoro ether) (fluorolink). The different blocks are highly immiscible with each other and form two-compartment micelles of mainly cylindrical shape in aqueous solution with lengths in the range of 100 to 200 nm and diameters of about 24 nm. The poly(perfluoro ether) (C blocks) forms the liquidlike center of the micelles (d = 6 nm). This is surrounded by a first shell of ca. 2 nm thickness consisting of beta-sheets of poly(gamma-benzyl l-glutamate) (B blocks) and a second 7 nm shell of poly(ethylene oxide) (A blocks). The A blocks provide water solubility, and the B and C blocks form separated hydrophobic compartments. This work is a contribution to the development of multicompartment micelles devoted to mimic transport proteins such as serum albumins in long-term development.

Random coils, beta-sheet ribbons, and alpha-helical fibers: one peptide adopting three different secondary structures at will.

To potentially cure neurodegenerative diseases, we need to understand on a molecular level what triggers the complex folding mechanisms and shifts the equilibrium from functional to pathological isoforms of proteins. The development of small peptide models that can serve as tools for such studies is of paramount importance. We describe the de novo design and characterization of an alpha-helical coiled coil based model peptide that contains structural elements of both alpha-helical folding and beta-sheet formation. Three distinct secondary structures can be induced at will by adjustment of pH or concentration. Low concentrations at pH 4.0 yield globular particles of the unfolded peptide, while at the same pH, but at higher concentration, defined beta-sheet ribbons are formed. In contrast, at high concentrations and pH 7.4, the peptide forms highly ordered alpha-helical fibers. Thus, this system allows one to systematically study now the consequences of the interplay between peptide and protein primary structure and environmental factors for peptide and protein folding on a molecular level.