Coatings are essential for biomedical applications antifouling and antimicrobial properties, supporting cell adhesion and tissue integration and particularly interesting in this field are nanogel ...(nGel)‐based coatings. Since biomaterials differ in physiochemical properties, specific nGel‐coating strategies need to be developed for every distinct material, leading to complex coating strategies. Hence, the solution lies in adopting a universal strategy to apply the same nGel coating with the same function on a wide range of implant surfaces. To this end, a universal nGel‐based coating approach provides the same coating using a single method on implant materials including stiff polymer materials, metals, ceramics, glass, and elastomers. The coating formation is achieved by electrostatic interactions between oxygen plasma–activated surfaces and positively charged nGels using a spray‐deposition method. Fluorescent labels are introduced into the nGels as a model for post‐modification capabilities to increase the functionality of the coating. The coating is highly stable under in vitro physiological conditions with the retention of its function on different clinically relevant materials. Meanwhile, the in vivo study indicates that the nGel coating on a polyvinylidene fluoride hernia mesh is stable and biocompatible, therefore, making the coating and the coating strategy, a highly impactful approach for future clinical developments.
The different classes of implants have diverse physicochemical properties, therefore it is essential to develop a nanogel‐coating approach that is translatable on every implant surface. The applied coating is homogenous and stable in both in vitro and in vivo conditions. This coating strategy indicates high potential for incorporation of different functionalities and is beneficial for advanced research in future.
In structural studies of immobilized, aggregated and self-assembled biomolecules, solid-state NMR (ssNMR) spectroscopy can provide valuable high-resolution structural information. Among the ...structural restraints provided by magic angle spinning (MAS) ssNMR the canonical focus is on inter-atomic distance measurements. In the current review, we examine the utility of ssNMR measurements of angular constraints, as a complement to distance-based structure determination. The focus is on direct measurements of angular restraints via the judicious recoupling of multiple anisotropic ssNMR parameters, such as dipolar couplings and chemical shift anisotropies. Recent applications are highlighted, with a focus on studies of nanocrystalline polypeptides, aggregated peptides and proteins, receptor-substrate interactions, and small molecule interactions with amyloid protein fibrils. The review also examines considerations of when and where ssNMR torsion angle experiments are (most) effective, and discusses challenges and opportunities for future applications.
The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington's disease. ...Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n*=4 for a simple, unbroken Q23 sequence to approximate unitary n* values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR 13C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn* values remain quite low (~10−10) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.
Display omitted
► β-Hairpin motifs added to short polyQ sequences greatly enhance aggregation rates. ► The enhancing effect is primarily due to a change in critical nucleus from 4 to 1. ► The amyloid structure of mutants is unchanged by solid-state NMR, electron microscopy, Fourier transform infrared spectroscopy, and bioactivities. ► Even with these added mutations, the monomeric polyQs are disordered in solution. ► The β-hairpin conformation is thus quite rare, consistent with a in nucleation.
Three zeolites (H‐Beta, H‐ZSM‐5 and H‐Y) were synthesized in the form of binder‐free macroscopic beads (d=215–840 μm) using a hydrothermal method employing anion‐exchange resin beads as hard ...template. The beads obtained after removal of the hard template by calcination consisted of crystalline zeolite domains connected with each other to form a hierarchical porous network in which the zeolitic micropores are accessible through meso‐ and macropores, as proven by characterization with XRD, N2 physisorption, SEM, and TEM. The composition, the nature and amount of acid sites and the degree of hydrophobicity of these beads were investigated by means of XRF, solid‐state NMR, pyridine‐FTIR and TGA. The zeolite beads were tested as heterogeneous catalysts in the Friedel‐Crafts acylation of anisole with acetic anhydride to produce para‐methoxyacetophenone. H‐Beta‐Beads displayed the best catalytic performance with 95 % conversion of acetic anhydride and 76 % yield of para‐methoxyacetophenone in a batch reactor test (90 °C, 6 h). Next, the catalytic performance of H‐Beta‐Beads was compared in both batch and continuous‐flow mode to extrudates prepared by mixing zeolite Beta powder with either kaolin or bentonite binders. H‐Beta‐Beads outperformed the extrudates in batch‐mode reactions and could be reused in multiple runs without discernible loss of activity. In the continuous‐flow test, H‐Beta‐Beads demonstrated higher average activity but deactivated more rapidly than the extrudates.
Friedel Crafts: Zeolite beads as catalysts for anisole acylation.
In this issue of Structure, Tang and colleagues probe how the Flemish mutation in amyloid precursor protein (APP) affects its conformation and cleavage by γ-secretase. They provide molecular insight ...into how an extracellular inhibitory element and cholesterol interactions affect the generation of Aβ peptides.
Display omitted
•Mutant huntingtin exon 1 fibrils feature a broad range of molecular dynamics.•Molecular motion is coupled to water dynamics outside the fiber core.•Dynamics-based spectral editing ...ssNMR reveals mobile non-core residues.•Intermediate-motion selection via dipolar dephasing of rigid sites.•Semi-mobile glutamines outside the fiber core observed and identified.
Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein’s expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils’ segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of ‘intermediate motion selection’ (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter’s detection with specific dyes and positron emission tomography tracers.
We show that multidimensional solid-state NMR 13C–13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only ...milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.
Display omitted
•Unprecedented sensitivity using recently developed AsymPol-POK hyperpolarizing agent.•Carbon-carbon correlation experiments at natural abundance on 1–2 mg of sample.•Examples on amyloid fibrils and microcrystals of pharmaceuticals at natural isotopic abundance.
Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to “misfolded” monomers. One way to vet these candidates is to ...develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary β-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called “βHP,” that exhibits greatly improved amyloid nucleation without measurably enhancing β-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within βHP. Modifications predicted to allow formation of a fully H-bonded β-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-β-hairpin H-bonding within βHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong β-hairpin encouraging motifs only rarely form β-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.
Display omitted
•β-Hairpin formation is implicated in polyQ amyloid nucleation and structure.•We prepared polyQ peptides combining β-hairpin motifs with backbone modifications.•Peptides with αN-Me-Gln or Pro mutations in the predicted hairpin aggregate poorly.•Such mutations at predicted non-H-bonding positions inhibit aggregation in trans.•The data clarify assembly mechanisms and provide valuable tools for disease studies.
We describe a cryogenic sample exchange system that dramatically improves the efficiency of magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments by reducing the time required to ...change samples and by improving long-term instrument stability. Changing samples in conventional cryogenic MAS DNP/NMR experiments involves warming the probe to room temperature, detaching all cryogenic, RF, and microwave connections, removing the probe from the magnet, replacing the sample, and reversing all the previous steps, with the entire cycle requiring a few hours. The sample exchange system described here—which relies on an eject pipe attached to the front of the MAS stator and a vacuum jacketed dewar with a bellowed hole—circumvents these procedures. To demonstrate the excellent sensitivity, resolution, and stability achieved with this quadruple resonance sample exchange probe, we have performed high precision distance measurements on the active site of the membrane protein bacteriorhodopsin. We also include a spectrum of the tripeptide N-
f-MLF-OH at 100
K which shows 30
Hz linewidths.
Alginate hydrogels are versatile self-assembling biocompatible materials with diverse biomedical and food industrial applications, which includes uses in encapsulation, (drug) delivery and tissue ...engineering. Hydrogel formation requires cross-linking, which for alginates is often done with calcium ions that engage in specific interactions with the polysaccharide carboxylic acid groups. Water molecules also hydrate these alginate groups and fill macropores within the hydrogels, with implications for both mechanical properties and cargo encapsulation. Understanding these aspects of hydrogels requires the observation and characterization of the hydrogel waters, how they engage the alginate, and fill the macropores. Here we employed solid-state NMR (ssNMR) spectroscopy to detect and study water molecules in re-hydrated alginate hydrogels. 1H, 2H, and 13C magic angle spinning (MAS) NMR and relaxation measurements were combined to observe both water and alginate. Two different water phases were detected that vary upon gradual (re)hydration of the alginate hydrogels. These water pools differ in their chemical shifts and NMR relaxation properties, reflecting hydration waters directly associated with the carbohydrate polymers alongside dynamic waters in the macropores. Thus, the ssNMR detects the water-filled macropore water pools and how they vary upon calcium cross-linking. We also observe how calcium cross-linking selectively immobilizes the α-guluronate monosaccharides, but leaves the β-mannuronate units more flexible and prone to selective re-hydration. Thus, these ssNMR experiments can be used to probe cross-linking and hydration of alginate hydrogels, with implications for our understanding of design parameters that tune their performance in (drug) delivery and other food industrial applications.
Display omitted
•Magic angle spinning NMR probed molecular aspects of alginate hydrogels.•Solid-state NMR used for detecting and quantifying water pools in hydrogels.•Solvent dynamics seen by NMR associate with distinct hydration water pools.•Impact of time-dependent cross-linking of alginate with calcium ions compared.•Molecular details of alginate dynamics, structure and re-hydration revealed.
PDF
