Designed amyloid beta peptide fibril - a tool for high-throughput screening of fibril inhibitors

Amyloid beta peptide (Abeta) fibril formation is widely believed to be the causative event of Alzheimer's disease pathogenesis. Therapeutic approaches are therefore in development that target various sites in the production and aggregation of Abeta. Herein we present a high-throughput screening tool to generate novel hit compounds that block Abeta fibril formation. This tool is an application for our fibril model (Abeta(16-37)Y(20)K(22)K(24))(4), which is a covalent assembly of four Abeta fragments. With this tool, screening studies are complete within one hour, as opposed to days with native Abeta(1-40). A Z' factor of 0.84+/-0.03 was determined for fibril formation and inhibition, followed by the reporter molecule thioflavin T. Herein we also describe the analysis of a broad range of reported inhibitors and non-inhibitors of Abeta fibril formation to test the validity of the system.     

Peptide‐Thiophene Hybrids as Self‐Assembling Conductive Hydrogels

A bottom‐up approach is taken to confer multidimensional structure to conductive polymers by attaching thiophene monomers to peptides predicted to self‐assemble into a biomimetic, fibrous nanostructure. A library of 12 peptides containing covalently attached thiophene monomers are synthesized. Peptide sequences capable of robust self‐assembly and hydrogel formation in aqueous media are further polymerized in situ and the physical and electrical properties are characterized. The resulting hybrid materials have conductivities in the range of 10^?2 to 10^?3 S cm^?1 and possess moduli in the range of several tissue types, making them potential candidates for use in tissue engineering and bioelectronic applications.     

Residue Folding Degree—Relationship to Secondary Structure Categories and Use as Collective Variable

Recently, we have shown that the residue folding degree, a network-based measure of folded content in proteins, is able to capture backbone conformational transitions related to the formation of secondary structures in molecular dynamics (MD) simulations. In this work, we focus primarily on developing a collective variable (CV) for MD based on this residue-bound parameter to be able to trace the evolution of secondary structure in segments of the protein. We show that this CV can do just that and that the related energy profiles (potentials of mean force, PMF) and transition barriers are comparable to those found by others for particular events in the folding process of the model mini protein Trp-cage. Hence, we conclude that the relative segment folding degree (the newly proposed CV) is a computationally viable option to gain insight into the formation of secondary structures in protein dynamics. We also show that this CV can be directly used as a measure of the amount of α-helical content in a selected segment.     

Cotranscriptionally Folded RNA Nanostructures Pave the Way to Intracellular Nanofabrication

A very attractive goal in nanotechnology is to manufacture smart nanodevices that integrate multiple biological/biomedical functions and autonomously function in vivo in a predefined and well‐controlled manner. For decades, researchers have been developing many different ways toward this target, using bottom‐up assembly of types of nanomaterials or top‐down fabrication of devices with nanometer‐scale precision. However, the practical application of these nanodevices remains challenging. One possible barrier lies in the spatiotemporal separation between fabrication and use, which poses a great challenge for the non‐invasive delivery of fully functional nanodevice into live cells. Indeed, cells themselves are highly complex natural machines with membrane barriers and finely regulated pathways for intracellular delivery. However, there is plenty of evidence that nanomaterials or nanodevices are easily aggregated or trapped inside of the cells.     

The composite nanomaterials containing (R)‐thalidomide‐molecularly imprinted polymers as a recognition system for enantioselective‐controlled release and targeted drug delivery

A molecularly imprinted polymer (MIP) enantioselective receptor for the (R)‐thalidomide enantiomer was synthesized and evaluated for its ability to deliver the drug to cancer cells. Polymer networks with precisely engineered binding sites were built into the assembled nanoparticles by a self‐organizing template in the prepolymerized mixture using methacrylic acid, a fluorescently active 2,6‐bis(acrylamido)pyridine and N,N′ methylene‐bis‐acrylamide, via both a covalent approach and a physical approach. The fine‐tuning of particle diameters was carried out by changes to the polymerizing synthesis method, the type of solvent and the amount of the poloxamer that led to an optimal formulation of the nanoparticles with sizes as small as 100 nm. Data from the ¹H‐nuclear magnetic resonance spectroscopy revealed the important structural motifs of an (R)‐thalidomide‐selective cavity for two different polymerization processes. We have investigated their ability for enantiomer recognition and the potential ability to protect the chiral MIP with a self‐assembled poloxamer structure. Moreover, the effect of the smaller molecular size can not only enable favorable imaging properties but also facilitate enhanced green fluorescence intensity for the deposited MIP and the (R)‐thalidomide in the poloxamer nanoparticles in a cell‐line in which the grafted MIP being higher than the deposited one. It was also demonstrated that the deposited MIP nanoparticles had the potential to make the drug effective for attacking multidrug ‐ resistant cells. Thus, the poloxamer nanoparticles containing a thermoresponsive MIP could maximize the release of the nontoxic (R)‐thalidomide at the tumor tissue, with the help of a proper temperature shift at the site. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41930.     

Applying γ‐Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

Rational choice of chemical modifications to proline residues allows the preorganization principle to be exploited for more stable assembly of the foldon domain as a tag for trimerization. With systematic knowledge of how chemical and steric variations of the ring substituents affect the relative stabilities of exo and endo puckers, the preorganization principle should then be usable in biotechnologically synthesized foldon mutants and applicable for protein tagging elsewhere.     

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.     

HMGA1a protein unfolds or refolds synthetic DNA-chromophore hybrid polymers: a chaperone-like behavior

High group mobility protein, HMGA1a, was found to play a chaperone-like role in the folding or unfolding of hybrid polymers that contained well-defined synthetic chromophores and DNA sequences. The synthetic and biological hybrid polymers folded into hydrophobic chromophoric nanostructures in water, but existed as partially unfolded configurations in pH or salt buffers. The presence of HMGA1a induced unfolding of the hybrid DNA-chromophore polymer in pure water, whereas the protein promoted refolding of the same polymer in various pH or salt buffers. The origin of the chaperone-like properties probably comes from the ability of HMGA1a to reversibly bind both synthetic chromophores and single stranded DNA. The unfolding mechanisms and the binding stoichiometry of protein-hybrid polymers depended on the sequence of the synthetic polymers.     

D-SAP: a new, noncytotoxic, and fully protease resistant cell-penetrating peptide

Protease resistant cell-penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self-assembling CPP-from all L-amino acids to all D-amino acids-and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self-assemble. The results of uptake experiments indicate that the protease-stable (that is, D-amino acid) analogue of the peptide is internalised by cells to the same extent as the protease-susceptible (that is, L-amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all-D analogue, which reveal the steps followed by the peptide upon its entry into the cell.     

Folding of Trp-cage Mini Protein Using Temperature and Biasing Potential Replica��Exchange Molecular Dynamics Simulations

The folding process of the 20 residue Trp-cage mini-protein was investigated using standard temperature replica exchange molecular dynamics (T-RexMD) simulation and a biasing potential RexMD (BP-RexMD) method. In contrast to several conventional molecular dynamics simulations, both RexMD methods sampled conformations close to the native structure after 10–20 ns simulation time as the dominant conformational states. In contrast, to T-RexMD involving 16 replicas the BP-RexMD method achieved very similar sampling results with only five replicas. The result indicates that the BP-RexMD method is well suited to study folding processes of proteins at a significantly smaller computational cost, compared to T-RexMD. Both RexMD methods sampled not only similar final states but also agreed on the sampling of intermediate conformations during Trp-cage folding. The analysis of the sampled potential energy contributions indicated that Trp-cage folding is favored by both van der Waals and to a lesser degree electrostatic contributions. Folding does not introduce any significant sterical strain as reflected by similar energy distributions of bonded energy terms (bond length, bond angle and dihedral angle) of folded and unfolded Trp-cage structures.     

Preparation and self‐assembly of polyaniline nanorods and their application as electroactive actuators

To improve the performance of ion‐exchange polymer–metal composite (IPMC) actuators, an electrical pathway material for enhancing the surface adhesion between the membrane and the metal electrodes of the IPMC was studied. As an efficient electrical pathway material, polyaniline nanorods (PANI‐NRs) doped with p‐toluene sulfonic acid (TSA) were synthesized with a template‐free method. The factors affecting polyaniline morphology were studied with various dopant concentrations and oxidant feeding rates. Highly conductive PANI‐NRs were formed when they were synthesized with ammonium persulfate at a 5.0 mL/min oxidant feeding rate and doped with 0.125M TSA. The conductivity of the PANI‐NRs was 1.15 × 10^?1 S/cm, and their diameters and lengths were 120–180 nm and 0.6–2 μm, respectively. To apply the membrane as an actuator, perfluorosulfonated ionomer (Nafion)/PANI‐NR blends were prepared by solution blending and casting. The actuating ability of the three‐layered membrane consisting of Nafion/PANI‐NR blends was then examined and compared with that of Nafion only. The actuating ability of the IPMC was improved when Nafion/PANI‐NRs were used as electrical pathways. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Structural insights into peptide self-assembly using photo-induced crosslinking experiments and discontinuous molecular dynamics

Determining the structure of the (oligomeric) intermediates that form during the self-assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo-induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid-forming peptide, Aβ_16-22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group—Fmoc(TFMD)Phe—was incorporated into the sequence (Aβ*_16–22). Electrospray ionization ion-mobility spectrometry mass-spectrometry (ESI-IMS-MS) analysis of the PIC products confirmed that Aβ*_16–22 forms assemblies with the monomers arranged as anti-parallel, in-register β-strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aβ_16-22 assembles through a single-step nucleation into a β-sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aβ_16-22 self-assembly processes, paving the way to explore the self-assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.     

In-cell solid-state NMR as a tool to study proteins in large complexes

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.     

Dendrimers: a review of their appeal and applications

Dendrimer chemistry is one of the most fascinating and rapidly expanding areas of modern chemistry. This review attempts to uncover the appeal of these structurally‐perfect branched macromolecules. The three intriguing regions of a dendritic molecule are discussed: the multifunctional surface, the tailored sanctuary within the branches, and the encapsulated core. The advantages of dendritic structures for property modification, and their potential applications in very diverse areas are illustrated. The exciting merger of dendrimer chemistry with self‐assembly offers new, stimulating avenues for exploration. Consequently, self‐assembling dendrimers comprise a major section of this article. © 2001 Society of Chemical Industry     

Layer-by-layer assembly of viral nanoparticles and polyelectrolytes: the film architecture is different for spheres versus rods

The development of tuneable thin film assemblies that contain (bio)nanoparticles is an emerging field in nanobiosciences/nanotechnology. Our research focuses on the utilisation of viral nanoparticles (VNPs) as tools and building blocks for materials science. In previous reports we studied multilayered arrays of chemically modified cowpea mosaic virus (CPMV) particles and linker molecules. To extend these studies and to gain more insights into the architecture of the arrays, we report here on the construction of multilayered assemblies of native plant viral particles and polyelectrolytes. We specifically addressed the question of whether the shape of the VNPs influences the overall structures of the arrays. To study this, we have chosen two particles with similar surface properties but different shapes: CPMV was used as a sphere-like VNP, and tobacco mosaic virus (TMV) served as a rod-shaped VNP. The multilayers were self-assembled on solid supports through electrostatic interactions. Multilayer build-up was followed by quartz crystal microbalance with dissipation monitoring and UV/Vis spectroscopy. Scanning electron microscopy was used to characterize the topologies of the thin films. Our studies show that shape indeed matters. Incorporation of CPMV in alternating arrays of VNPs and polyelectrolytes is demonstrated; in stark contrast, TMV particles were found to be excluded from the arrays, and floated atop the architecture in an ordered structure.     

Preparation of a three‐dimensional ordered macroporous titanium dioxide material with polystyrene colloid crystal as a template

Polystyrene (PS) photonic colloid crystals were assembled from PS spheres prepared by emulsion‐free polymerization through an improved vertical deposition method that could shorten the assembly time efficiently. The monodispersity of the spheres was appraised according to the standard deviation. The results showed that the PS spheres had a high monodispersity with a standard deviation of 3.7% and a dispersion coefficient of 0.02. The morphology and bandgap structure were observed with scanning electron microscopy images and transmission spectra, respectively. The mechanism of vertical deposition was analyzed simply. As an application of PS colloid crystals, ordered macroporous TiO₂ photonic crystals were prepared, and the structure and properties of macroporous TiO₂ were also studied with various analytical methods, which provided some values for the fabrication of photonic crystals with a complete bandgap. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

From Side Chains Rattling on Picoseconds to Ensemble Simulations of Protein Folding

Simulations of biological macromolecules have evolved tremendously since the discoveries of the 1970s. The field has moved from simple simulations in vacuo on picosecond scales to milliseconds of accurate sampling of large proteins, and it has become a standard tool in biochemistry and biophysics, rather than a dedicated theoretical one. This is partly due to increasing computational power, but it would not have been possible without huge research efforts invested in new algorithms and software. Here, we illustrate some of this development, both past and future challenges, and in particular, discuss how the recent introduction of modern ensemble methods is breaking the trend of ever-longer simulations to instead focus on throughput and sampling. This has not only helped simulations become much more accurate, but it provides statistical error estimates, which are critical, as simulations are increasingly used to predict properties that have not yet been measured experimentally.