Disulfide bonds between Cys residues in adjacent strands of parallel β-sheets are rare among proteins, which suggests that parallel β-sheet structure is not stabilized by such disulfide cross-links. ...We report experimental results that show, surprisingly, that an interstrand disulfide bond can stabilize parallel β-sheets formed by an autonomously folding peptide in aqueous solution. NMR analysis reveals that parallel β-sheet structure is terminated beyond the disulfide bond, which causes deviation from the extended backbone conformation at one of the Cys residues.
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IJS, KILJ, NUK, PNG, UL, UM
Proteinogenic amino acid residues that promote β-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display β-sheet ...secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also bear an ionizable group in the side chain.
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A highly stereoselective synthesis of novel cyclically constrained γ-amino acid residues is presented. The key step involves organocatalytic Michael addition of an aldehyde to 1-nitrocyclohexene. ...After aldehyde reduction, this approach provides optically active β-substituted δ-nitro alcohols (96−99% ee), which can be converted to γ-amino acid residues with a variety of substituents at the α position. We have used these new building blocks to prepare α/γ-peptide foldamers that adopt a specific helical conformation in solution and in the solid state.
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Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. ...Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly 13C18O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm–1, with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm–1 line width. Narrower line widths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel β-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of β-sheet containing proteins and protein assemblies, such as amyloid fibers.
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We report the first high-resolution structural data for the β/γ-peptide 13-helix (i,i+3 CO···HN H-bonds), a secondary structure that is formed by oligomers with a 1:1 alternation of β- and γ-amino ...acid residues. Our characterization includes both crystallophaphic and 2D NMR data. Previous studies suggested that β/γ-peptides constructed from conformationally flexible residues adopt a different helical secondary structure in solution. Our design features preorganized β- and γ-residues, which strongly promote 13-helical folding by the 1:1 β/γ backbone.
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We report the asymmetric synthesis of the γ-amino acid (1R,2R)-2-aminomethyl-1-cyclopentane carboxylic acid (AMCP) and an evaluation of this residue’s potential to promote secondary structure in ...α/γ-peptides. Simulated annealing calculations using NMR-derived distance restraints obtained for α/γ-peptides in chloroform reveal that AMCP-containing oligomers are conformationally flexible. However, additional evidence suggests that an internally hydrogen-bonded helical conformation is partially populated in solution. From these data, we propose characteristic NOE patterns for the formation of the α/γ-peptide 12/10-helix and discuss the apparent conformational frustration of AMCP-containing oligomers.
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Molecular design can be used to investigate whether the parallel‐β‐sheet secondary structure is subject to length‐dependent cooperative stabilization along the strand direction (see picture). ...Extending strands by adding high‐propensity residues (B) leads to a steady increase in parallel‐β‐sheet stability, but inclusion of low‐propensity residues (A) can negate this stabilization.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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•Opuntia ficus-indica cladode extract (CE) is a phagostimulant to Spodoptera frugiperda caterpillars.•CE disorganized the midgut epithelium and increased cytoplasm vacuolation of ...columnar cells of caterpillars.•CE increased the size of goblet cells and induced the disappearance of the peritrophic membrane at the midgut of caterpillars.•CE reduced the amount of neutral polysaccharides and protein content at the midgut epithelial cells of caterpillars.
The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous insect whose control mainly involves the use of synthetic insecticides that can be highly toxic to non-target organisms and lead to the selection of resistant populations. This work reports the effects of the saline extract of Opuntia ficus-indica cladodes (CE) on the feeding survival and midgut morphophysiology of S. frugiperda third instar caterpillars. CE exerted a phagostimulant effect to the caterpillars but was not able to kill them. Despite of this, the midguts of caterpillars treated with CE (12.8 mg/g) showed intense disorganization of epithelium, vacuolation of the cytoplasm of columnar cells and increased size of goblet cells, as well as absence of the peritrophic membrane. The caterpillars treated with CE showed a reduction in the amount of neutral polysaccharides and protein content at the midgut epithelial cells. Together these data may indicate that the ingestion of CE in higher concentrations or by a longer period would lead the caterpillars to death or to damage in development. In conclusion, CE ingestion resulted in damage to the midgut morphology of caterpillars, with a reduction in carbohydrate and protein content.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent advancements in cell culture engineering have allowed drug manufacturers to achieve higher productivity by driving higher product titers through cell line engineering and high‐cell densities. ...However, these advancements have shifted the burden to clarification and downstream processing where the difficulties now revolve around removing higher levels of process‐ and product‐related impurities. As a result, a lot of research efforts have turned to developing new approaches and technologies or process optimization to still deliver high quality biological products while controlling cost of goods. Here, we explored the impact of a novel single use technology employing chromatographic principle‐based clarification for a process‐intensified cell line technology. In this study, a 16% economic benefit ($/g) was observed using a single‐use chromatographic clarification compared to traditional single‐use clarification technology by improving the overall product cost through decreased operational complexity, higher loading capacity, increased product recovery, and higher impurity clearance. In the end, the described novel chromatographic approach significantly simplified and enhanced the cell culture fluid harvest unit operation by combining the reduction of insoluble and key soluble contaminants of the harvest fluid into a single stage.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most ...patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model.
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