Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer’s disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2–R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When β-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel β-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn-binding histidines with binding geometries unusual in proteins. Each Zn has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn.

There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens that are resistant to most antibiotics. We have analyzed their structure-function relationship and antimicrobial mechanisms using membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.

We report the identification of a new class of antimicrobial peptidomimetics-γ-AApeptides with potent and broad-spectrum activity, including clinically-relevant strains that are unresponsive to most antibiotics. They are also not prone to select for drug-resistance.

A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFPbased fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila.Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.