775 results on '"Trifluoroethanol"'
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2. The Effect of Trifluoroethanol and Glycerol on the Thermal Properties of Collagen Using Optical Displacement-Enhanced Heterodyne Polarimeter
- Author
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Chien-Ming Wu, Horn-Haw Chen, Kai-Han Tseng, and Hung-Wei Chen
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collagen protection ,heterodyne polarimeter ,thermal denaturation ,trifluoroethanol ,glycerol ,Technology ,Engineering (General). Civil engineering (General) ,TA1-2040 ,Biology (General) ,QH301-705.5 ,Physics ,QC1-999 ,Chemistry ,QD1-999 - Abstract
An angular displacement-enhanced heterodyne polarimeter has been employed to investigate the interplay between trifluoroethanol (TFE) and glycerol on the thermal denaturation of type I collagen. The concentration of the collagen solution was fixed at 0.341 (mg/mL), and was heated from 25 °C to 55 °C. TFE solutions with concentrations of 5%, 10%, 15%, 20%, 40% and 80% (v/v) were prepared and the phase change was recorded for the determination of thermal denaturation. It was observed that the thermal denaturation temperature (Td) is decreased with increasing TFE concentration due to the partial cleavage of the triple-helical structure. With TFE concentration higher than 20% (v/v), the degree of optical rotation appears to be nearly the same, reflecting that the collagen triple helices have been completely destructed. Moreover, the addition of glycerol in inhibiting the thermal denaturation of collagen is investigated. It has been shown that glycerol can improve the thermal denaturation of both collagen and TFE-mixed collagen. Experimental results show that, in the presence of 2 M glycerol, the Td of collagen remained at around 41.9 °C, meanwhile the Td of 20% (v/v) TFE-mixed collagen is significantly restored to 32.8 °C.
- Published
- 2015
- Full Text
- View/download PDF
3. Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca
- Author
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P.B. Timmons and Chandralal M. Hewage
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Protein Conformation, alpha-Helical ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Science ,Static Electricity ,Antineoplastic Agents ,Peptide ,Molecular Dynamics Simulation ,Micelle ,Article ,Molecular dynamics ,chemistry.chemical_compound ,Amphiphile ,Amino Acid Sequence ,Sodium dodecyl sulfate ,Peptide sequence ,Micelles ,chemistry.chemical_classification ,Membranes ,Multidisciplinary ,Cationic polymerization ,Sodium Dodecyl Sulfate ,Water ,Trifluoroethanol ,Nuclear magnetic resonance spectroscopy ,Antimicrobial ,Anti-Bacterial Agents ,chemistry ,Medicine ,Molecular modelling ,Hydrophobic and Hydrophilic Interactions ,Solution-state NMR ,Antimicrobial Cationic Peptides - Abstract
Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d_{3}$$\end{document}d3 mixture. The structure is defined by an \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document}α-helix that spans between Ile\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{6}$$\end{document}6-Ala\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{26}$$\end{document}26, and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide’s interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document}α-helical conformation between residues Ile\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{6}$$\end{document}6-Ala\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{26}$$\end{document}26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.
- Published
- 2021
4. Correlating solvation with conformational pathways of proteins in alcohol–water mixtures: a THz spectroscopic insight
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Rajib Kumar Mitra, Himanshu Gohil, S. S. Prabhu, Debasish Das Mahanta, and Partha Pyne
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Terahertz Spectroscopy ,Circular dichroism ,Aqueous solution ,Ethanol ,Protein Conformation ,Terahertz radiation ,Solvation ,Water ,General Physics and Astronomy ,Alcohol ,Lactoglobulins ,Trifluoroethanol ,Protein Structure, Secondary ,chemistry.chemical_compound ,Solubility ,chemistry ,Chemical physics ,Attenuation coefficient ,Biophysical Process ,Animals ,Cattle ,Muramidase ,Physical and Theoretical Chemistry ,Absorption (chemistry) ,Chickens - Abstract
Water, being an active participant in most of the biophysical processes, is important to trace how protein solvation changes as its conformation evolves in the presence of solutes or co-solvents. In this study, we investigate how the secondary structures of two diverse proteins - lysozyme and β-lactoglobulin - change in the aqueous mixtures of two alcohols - ethanol and 2,2,2-trifluoroethanol (TFE) using circular dichroism measurements. We observe that these alcohols change the secondary structures of these proteins and the changes are protein-specific. Subsequently, we measure the collective solvation dynamics of these two proteins both in the absence and in the presence of alcohols by measuring the frequency-dependent absorption coefficient (α(ν)) in the THz (0.1-1.2 THz) frequency domain. The alcohol-water mixtures exhibit a non-ideal behaviour with the highest absorption difference (Δα) obtained at Xalcohol = 0.2. The protein solvation in the presence of the alcohols shows an oscillating behaviour in which Δαprotein changes with Xalcohol. Such an oscillatory behaviour of protein solvation results from a delicate interplay between the protein-water, protein-alcohol and water-alcohol associations. We attempt to correlate the various structural conformations of the proteins with the associated solvation.
- Published
- 2021
5. Trifluoroethanol Modulates Amyloid Formation by the All α-Helical URN1 FF Domain
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Patrizia Marinelli, Salvador Ventura, and Virginia Castillo
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α-helix ,amyloid ,FF domain ,trifluoroethanol ,molten globule ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Amyloid fibril formation is implicated in different human diseases. The transition between native α-helices and nonnative intermolecular β-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small all-α protein that populates a molten globule (MG) at low pH. Despite the fact that this conformation maintains most of the domain native secondary structure, it progressively converts into β-sheet enriched and highly ordered amyloid fibrils. In this study, we investigated if 2,2,2-trifluoroethanol (TFE) induced conformational changes that affect URN1-FF amyloid formation. Despite TFE having been shown to induce or increase the aggregation of both globular and disordered proteins at moderate concentrations, we demonstrate here that in the case of URN1-FF it reinforces its intrinsic α-helical structure, which competes the formation of aggregated assemblies. In addition, we show that TFE induces conformational diversity in URN1-FF fibrils, in such a way that the fibrils formed in the presence and absence of the cosolvent represent different polymorphs. It is suggested that the effect of TFE on both the soluble and aggregated states of URN1-FF depends on its ability to facilitate hydrogen bonding.
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- 2013
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6. Trifluoroethanol Partially Unfolds G93A SOD1 Leading to Protein Aggregation: A Study by Native Mass Spectrometry and FPOP Protein Footprinting
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Jill A. Zitzewitz, Michael L. Gross, Ben Niu, C. Robert Matthews, and Brian C. Mackness
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Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Protein Conformation ,animal diseases ,Electrospray ionization ,Mutant ,SOD1 ,Protein aggregation ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,Article ,Protein Aggregates ,03 medical and health sciences ,chemistry.chemical_compound ,Humans ,Protein Footprinting ,Protein Unfolding ,0303 health sciences ,Superoxide Dismutase ,Protein footprinting ,030302 biochemistry & molecular biology ,nutritional and metabolic diseases ,Intact protein ,Trifluoroethanol ,nervous system diseases ,Monomer ,chemistry ,Biophysics - Abstract
Misfolding of Cu, Zn superoxide dismutase (SOD1) variants may lead to protein aggregation and ultimately amyotrophic lateral sclerosis (ALS). The mechanism and protein conformational changes during this process are complex and remain unclear. To study SOD1 variants aggregation at the molecular level and in solution, we chemically induced aggregation of a mutant variant (G93A SOD1) with trifluoroethanol (TFE) and used both native mass spectrometry (MS) to analyze the intact protein and Fast Photochemical Oxidation of Proteins (FPOP) to characterize the structural changes induced by TFE. We found partially unfolded G93A SOD1 monomers prior to oligomerization and identified regions of the N-terminus, C-terminus, and strands β5, β6 accountable for the partial unfolding. We propose that exposure of hydrophobic interfaces of these unstructured regions serves as a precursor to aggregation. Our results provide a possible mechanism and molecular basis for ALS-linked SOD1 misfolding and aggregation.
- Published
- 2020
7. Direct transfer of tri- and di-fluoroethanol units enabled by radical activation of organosilicon reagents
- Author
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Zhihong Zhu, Weilu Zhang, Gang Zhou, Xiao Shen, Zhengyu Li, Shanshan Liu, Xingxing Gong, and Xiang Chen
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Alkylation ,Reaction mechanisms ,Science ,General Physics and Astronomy ,Synthetic chemistry methodology ,Chemistry Techniques, Synthetic ,Direct transfer ,010402 general chemistry ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,Article ,chemistry.chemical_compound ,Molecule ,Organosilicon Compounds ,lcsh:Science ,Organosilicon ,Antitumor activity ,Multidisciplinary ,Ethanol ,Molecular Structure ,010405 organic chemistry ,General Chemistry ,Trifluoroethanol ,Homogeneous catalysis ,Combinatorial chemistry ,0104 chemical sciences ,chemistry ,Intramolecular force ,Reagent ,Alcohols ,Alkoxyl radicals ,Indicators and Reagents ,lcsh:Q - Abstract
Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z′., Methods for direct incorporation of tri- and di-fluoroethanol units in molecules are relatively limited. Here, the authors report two organosilicon reagents which are applied to allylation, alkylation and alkenylation reactions as tri- and di-fluoroethanol transfer reagents.
- Published
- 2020
8. Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C‐Terminal Domain of Histone H1.0
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Belén Chaves-Arquero, José Manuel Pérez-Cañadillas, M. Angeles Jiménez, Ministerio de Economía y Competitividad (España), Consejo Superior de Investigaciones Científicas (España), and Ministerio de Ciencia, Innovación y Universidades (España)
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Magnetic Resonance Spectroscopy ,Stereochemistry ,010402 general chemistry ,Intrinsically disordered proteins ,01 natural sciences ,Catalysis ,Structureelucidation ,Histones ,Protein models ,NMR spectroscopy ,Protein structure ,Histone H1 ,310 helix ,Side chain ,Humans ,Phosphorylation ,010405 organic chemistry ,Chemistry ,Structure elucidation ,C-terminus ,Organic Chemistry ,Trifluoroethanol ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Proteinstructures ,0104 chemical sciences ,Intrinsically Disordered Proteins ,Peptides - Abstract
12 pags., 7 figs., 3 tabs., o investigate the structural impact of phosphory-lation on the human histone H1.0 C-terminal domain,weperformed NMR structural studies of model peptides con-taining asingle phosphorylation site:T118-H1.0 (T118PKKmotif) andT140-H1.0 (T140PVK motif). Both model peptides aremainly disordered in aqueous solution in their non-phos-phorylated and phosphorylated forms, but become struc-tured in the presence of trifluoroethanol. The peptides T118-H1.0 and pT118-H1.0 contain two helical regions, along am-phipathic a helix spanning residues 104–115 and ashort a/310helix (residues 119–123),that are almostperpendicular inT118-H1.0 but have apoorly defined orientation in pT118-H1.0.Peptides T140-H1.0 and pT140-H1.0 form very similar a helicesbetween residues 141–147. The TPKK and TPVK motifs showthe same backbone conformation,but differ in their side-chain contacts;the Thr and pThr side chains interact withthe i + 2Lys side chain in the TPKK motif, andwith the i+ 3Lysside chain in the TPVK motif. The pT phosphate group inpT118-H1.0andpT140-H1.0haspKavaluesbelowtheintrinsicvalues, which can be explained by non-specific charge–chargeinteractions with nearbyLys. The non-polar ValintheTPVK motif accounts forthe pT140pKabeing closer to the in-trinsic pKavalue than the pT118pKa.Altogether,these resultsvalidate that minimalist strategies using model peptides canprovide structural details difficult to obtain in short-lived in-trinsically disordered proteins and domains., We thank the financial support from projects no. CTQ2014-52633-P (MINECO of Spain) and CTQ2017-84371-P (Spanish Agencia Estatal de Investigacin /Fondo Europeo de Desarrollo Regional;AEI/FEDER of the EU). B.B. was a recipient of a predoctoral FPI scholarship (BES-2015-073383)from MINECO. The NMR experiments were performed in the “Manuel Rico” NMR laboratory,LMR, CSIC,part of the SpanishLarge-Scale National Facility ICTS R-LRB
- Published
- 2020
9. Biomacromolecules enabled dendrite-free lithium metal battery and its origin revealed by cryo-electron microscopy
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Yujing Liu, Chengbin Jin, Xinyong Tao, Zhong Jin, Tiefeng Liu, Ouwei Sheng, Zhijin Ju, Zhang Wenkui, Huadong Yuan, Jianwei Nai, He Tian, Yao Wang, and Jianhui Zheng
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Biomineralization ,Battery (electricity) ,Energy storage ,Materials science ,Macromolecular Substances ,Science ,General Physics and Astronomy ,chemistry.chemical_element ,Nanotechnology ,02 engineering and technology ,Electrolyte ,Lithium ,010402 general chemistry ,01 natural sciences ,Article ,General Biochemistry, Genetics and Molecular Biology ,Egg Shell ,Batteries ,Electric Power Supplies ,Animals ,lcsh:Science ,Electrodes ,Multidisciplinary ,Cryoelectron Microscopy ,Electrochemical Techniques ,Trifluoroethanol ,General Chemistry ,Chemical Engineering ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Anode ,Membrane ,chemistry ,Electrode ,lcsh:Q ,Dendrite (metal) ,Crystallization ,0210 nano-technology - Abstract
Metallic lithium anodes are highly promising for revolutionizing current rechargeable batteries because of their ultrahigh energy density. However, the application of lithium metal batteries is considerably impeded by lithium dendrite growth. Here, a biomacromolecule matrix obtained from the natural membrane of eggshell is introduced to control lithium growth and the mechanism is motivated by how living organisms regulate the orientation of inorganic crystals in biomineralization. Specifically, cryo-electron microscopy is utilized to probe the structure of lithium at the atomic level. The dendrites growing along the preferred crystallographic orientation are greatly suppressed in the presence of the biomacromolecule. Furthermore, the naturally soluble chemical species in the biomacromolecules can participate in the formation of solid electrolyte interphase upon cycling, thus effectively homogenizing the lithium deposition. The lithium anodes employing bioinspired design exhibit enhanced cycling capability. This work sheds light on identifying substantial challenges in lithium anodes for developing advanced batteries., Inspired by the role of proteins in regulating eggshell mineralization, here Tao, Liu and colleagues apply trifluoroethanol modified eggshell membrane to combat lithium dendrite. Cryo-electron microscopy reveals that the growth along the most favored crystallographic direction is suppressed.
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- 2020
10. Synthesis of Some New Fluorinated Hexahydroquinoline and Acridinedione Derivatives in Trifluoroethanol
- Author
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Cosmas O. Okoro, Mumiye A. Ogunwale, and Tasneem Siddiquee
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unsymmetric Hantzsch reaction ,dihydropyridine ,trifluoromethyl ,hexahydroquinoline ,acridinedione ,trifluoroethanol ,Technology ,Engineering (General). Civil engineering (General) ,TA1-2040 ,Biology (General) ,QH301-705.5 ,Physics ,QC1-999 ,Chemistry ,QD1-999 - Abstract
This article describes one-pot synthesis of new fluorinated hexahydroquinoline derivatives via unsymmetric Hantzsch reaction involving 5-trifluoromethyl-1,3-cyclohexanedione, aldehydes, acetoacetate ester, and ammonium acetate in trifluoroethanol (TFE). The reaction is simple and rapid with high yield.
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- 2012
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11. Solubilization and refolding of variety of inclusion body proteins using a novel formulation
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Rahul Ahuja, Priyank Singhvi, Ankit Saneja, and Amulya K. Panda
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Inclusion Bodies ,Functional protein ,Chemistry ,Human Growth Hormone ,Human growth hormone ,Industrial scale ,Proteins ,General Medicine ,Trifluoroethanol ,Protein aggregation ,Biochemistry ,Protein tertiary structure ,Inclusion bodies ,Protein Refolding ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Solubility ,Structural Biology ,Solubilization ,Escherichia coli ,Humans ,Molecular Biology ,Protein secondary structure - Abstract
Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many mild solubilization buffers in last two decades, high-throughput recovery of functional protein from wide range of IBs is still a challenge at an academic and industrial scale. Herein, a novel formulation for improved recovery of bioactive protein from variety of bacterial IBs is developed. This novel formulation is comprised of 20% trifluoroethanol, 20% n-propanol and 2 M urea at pH 12.5 which disrupts the major dominant forces involved in protein aggregation. An extensive comparative study of novel formulation conducted on different IBs demonstrates its high solubilization and refolding efficiency. The overall yield of bioactive protein from human growth hormone expressed as bacterial IBs is reported to be around 50%. This is attributed to the capability of novel formulation to disrupt the tertiary structure of the protein while protecting the secondary structure of the protein, thereby reducing the formation of soluble aggregates during refolding. Thus, the formulation can eliminate the need of screening and optimizing various solubilization formulation and will improve the efficiency of recovering bioactive protein from variety of IB aggregates.
- Published
- 2021
12. The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice
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Mayuko Fukuoka, Midori Nohara, Yoshihiro Kanamarua, Xijier, Tomio Yabe, Chihiro Kobayashi, and Mizuho Inagaki
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Diarrhea ,Protein Denaturation ,Cellular differentiation ,Cell ,Crypt ,Lactoglobulins ,Microbiology ,Cell Line ,03 medical and health sciences ,Mice ,medicine ,Ingestion ,Animals ,Humans ,Cytotoxicity ,Barrier function ,030304 developmental biology ,Cell Proliferation ,0303 health sciences ,Mice, Inbred BALB C ,Cell growth ,Chemistry ,030302 biochemistry & molecular biology ,food and beverages ,Cell Differentiation ,General Medicine ,Trifluoroethanol ,Animals, Suckling ,Gastrointestinal Tract ,medicine.anatomical_structure ,Whey Proteins ,Caco-2 ,Lactalbumin ,Animal Science and Zoology ,Cattle ,Caco-2 Cells ,Food Science - Abstract
Alpha-lactalbumin (α-LA) and β-lactoglobulin (β-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and β-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and β-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.
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- 2021
13. The antimicrobial peptides casocidins I and II: Solution structural studies in water and different membrane-mimetic environments
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Simonetta Caira, Flavia Anna Mercurio, Andrea Scaloni, and Marilisa Leone
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casocidins ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Physiology ,Antimicrobial peptides ,030209 endocrinology & metabolism ,Phosphatidylserines ,Cleavage (embryo) ,Biochemistry ,TFE ,antimicrobial peptides ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Endocrinology ,conformational switch ,Chemistry ,Vesicle ,Bilayer ,Caseins ,Water ,Trifluoroethanol ,Nuclear magnetic resonance spectroscopy ,NMR ,Peptide Fragments ,lipid vesicles ,Peptide Conformation ,Membrane ,Cytoplasm ,Phosphatidylcholines ,Biophysics ,030217 neurology & neurosurgery ,Antimicrobial Cationic Peptides - Abstract
Antimicrobial peptides (AMPs) represent crucial components of the natural immune defense machinery of different organisms. Generally, they are short and positively charged, and bind to and destabilize bacterial cytoplasmic membranes, ultimately leading to cell death. Natural proteolytic cleavage of ?s2-casein in bovine milk generates the antimicrobial peptides casocidin I and II. In the current study, we report for the first time on a detailed structure characterization of casocidins in solution by means of Nuclear Magnetic Resonance spectroscopy (NMR). Structural studies were conducted in H2O and different membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE) and lipid anionic and zwitterionic vesicles. For both peptides, results indicate a mainly disordered conformation in H2O, with a few residues in a partial helical structure. No wide increase of order occurs upon interaction with lipid vesicles. Conversely, peptide conformation becomes highly ordered in presence of TFE, with both casocidins presenting a large helical content. Our data point out a preference of casocidins to interact with model anionic membranes. These results are compatible with possible mechanisms of action underlying the antimicrobial activity of casocidins that ultimately may affect membrane bilayer stability.
- Published
- 2019
14. Examination of Trifluoroethanol Interactions with Trp-Cage in Trifluoroethanol–Water at 298 K through Molecular Dynamics Simulations and Intermolecular Nuclear Overhauser Effects
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John T. Gerig
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Molecular Conformation ,chemistry.chemical_element ,Peptide ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Diffusion ,Molecular dynamics ,Computational chemistry ,0103 physical sciences ,Materials Chemistry ,Physical and Theoretical Chemistry ,chemistry.chemical_classification ,010304 chemical physics ,Chemistry ,Intermolecular force ,Temperature ,Tryptophan ,Water ,Hydrogels ,Trifluoroethanol ,0104 chemical sciences ,Surfaces, Coatings and Films ,Solvents ,Fluorine ,Protein model ,Cage - Abstract
Molecular dynamics simulations of the protein model Trp-cage in 42% trifluoroethanol (TFE)-water at 298 K have been carried out with the goal of exploring peptide hydrogen-solvent fluorine nuclear spin cross-relaxation. The TFE5 model of TFE developed in a previous work was used with the TIP5P-Ew model of water. System densities and component translational diffusion coefficients predicted by the simulations were within 20% of the experimental values. Consideration of the calculated relative amounts of TFE and water surrounding the hydrogens of Trp-cage indicated that the composition of the solvent mixture beyond ∼1.5 nm from the van der Waals surface of the peptide is close to the composition of the bulk solvent, but as observed by others, TFE accumulates preferentially near the peptide surface. In the simulations, both TFE and water molecules make contacts with the peptide surface; water molecules predominate in contacts with the peptide backbone atoms and TFE molecules generally preferentially interact with side chains. Translational diffusion of solvent molecules appears to be slowed near the surface of the peptide. Depending on the location in the structure, TFE molecules form complexes with the peptide that may persist for up to ∼7 ns. Many of the peptide spin-solvent fluorine cross-relaxation parameters (Σ
- Published
- 2019
15. Two different regimes in alcohol-induced coil-helix transition: effects of 2,2,2-trifluoroethanol on proteins being either independent of or enhanced by solvent structural fluctuations
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Tomonari Sumi, Takeshi Morita, Hiroyo Ohgi, Yoshikata Koga, Hiroshi Imamura, Peter Westh, and Keiko Nishikawa
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Molecular Conformation ,General Physics and Astronomy ,010402 general chemistry ,01 natural sciences ,Melittin ,Phase Transition ,03 medical and health sciences ,chemistry.chemical_compound ,Molecule ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Aqueous solution ,Biomolecule ,Water ,Trifluoroethanol ,Melitten ,0104 chemical sciences ,Solvent ,Crystallography ,chemistry ,2,2,2-Trifluoroethanol ,Alcohols ,Helix ,Solvents ,Thermodynamics ,Methanol ,Peptides - Abstract
Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifluoroethanol (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by small-angle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol 2O mixture is unexpectedly comparable to those in the series of mono-ols. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below ∼2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above ∼2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosolvent effects of TFE on proteins/peptides.
- Published
- 2021
16. Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity
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Jing-Ting Chiou, Yi-Jun Shi, Liang-Jun Wang, Yuan-Chin Lee, Long-Sen Chang, and Chia-Hui Huang
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Cell Membrane Permeability ,Membrane permeability ,02 engineering and technology ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Cell Line, Tumor ,Animals ,Humans ,Molecular Biology ,Protein secondary structure ,030304 developmental biology ,Lactalbumin ,0303 health sciences ,Semicarbazide ,Quenching (fluorescence) ,Chemistry ,Cytotoxins ,Circular Dichroism ,Cell Membrane ,General Medicine ,Trifluoroethanol ,U937 Cells ,021001 nanoscience & nanotechnology ,Molten globule ,Protein tertiary structure ,Protein Structure, Tertiary ,Membrane ,Biophysics ,Cattle ,0210 nano-technology - Abstract
We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.
- Published
- 2020
17. Protein environment: a crucial triggering factor in Josephin domain aggregation : the role of 2,2,2-trifluoroethanol
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Paolo Tortora, Gianvito Grasso, Salvador Ventura, Marco Agostino Deriu, Susanna Navarro, Cristina Visentin, Maria Elena Regonesi, Visentin, C, Navarro, S, Grasso, G, Regonesi, M, Deriu, M, Tortora, P, and Ventura, S
- Subjects
0301 basic medicine ,Secondary ,Circular dichroism ,Molecular dynamic ,Protein Conformation ,Molecular Conformation ,Protein aggregation ,Josephin domain ,01 natural sciences ,Protein Structure, Secondary ,lcsh:Chemistry ,Molecular dynamics ,chemistry.chemical_compound ,Protein structure ,Ataxin-3 ,lcsh:QH301-705.5 ,Spectroscopy ,Amyloid aggregation ,Protein Stability ,Circular Dichroism ,General Medicine ,Computer Science Applications ,2,2,2-trifluoroethanol ,Protein-cosolvent interaction ,Amyloid ,Humans ,Molecular Dynamics Simulation ,Peptides ,Protein Aggregates ,Protein Domains ,Protein Structure, Tertiary ,Repressor Proteins ,Trifluoroethanol ,2,2,2-Trifluoroethanol ,Peptide ,Spinocerebellar ataxia ,Human ,Protein Structure ,Protein Domain ,Protein domain ,010402 general chemistry ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,medicine ,Physical and Theoretical Chemistry ,Molecular Biology ,Organic Chemistry ,Repressor Protein ,medicine.disease ,molecular dynamics ,0104 chemical sciences ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,2-trifluoroethanol ,Biophysics ,Protein Aggregate ,Tertiary - Abstract
The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially &alpha, helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.
- Published
- 2020
18. Sol-gel preparation of fluorine-containing hydrophobic SiO2 coating by using trifluoroethanol as dispersant and modifier simultaneously
- Author
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Mengmeng Zhu, Longqiang Ye, Shuang Cai, Liang Jiang, and Yulu Zhang
- Subjects
Materials science ,General Physics and Astronomy ,02 engineering and technology ,engineering.material ,01 natural sciences ,Dispersant ,Contact angle ,Coating ,X-ray photoelectron spectroscopy ,0103 physical sciences ,Fourier transform infrared spectroscopy ,Alkyl ,Sol-gel ,010302 applied physics ,chemistry.chemical_classification ,SiO2 coating ,Fluorine containing ,Trifluoroethanol ,021001 nanoscience & nanotechnology ,Hydrophobic ,lcsh:QC1-999 ,chemistry ,Chemical engineering ,engineering ,0210 nano-technology ,Sol-gel method ,lcsh:Physics - Abstract
A facile sol-gel method was proposed to prepare fluorine-containing hydrophobic SiO2 coating using trifluoroethanol (TFE) as the dispersant and hydrophobic modifier simultaneously. FTIR and XPS analysis indicated that CF3 groups were incorporated onto the surface of SiO2 particles. The resultant SiO2 coating possessed excellent hydrophobicity with a water contact angle (WCA) of 145°. This provides a new method for the preparation of fluorine-containing sol-gel SiO2 coating without the expensive long-chain alkyl fluorosilane.
- Published
- 2020
19. Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations
- Author
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Prateek Kumar, Rajanish Giri, Shobha Kumari, Amit Kumar, and Vladimir N. Uversky
- Subjects
0301 basic medicine ,Protein Folding ,Biophysics ,Peptide ,Molecular Dynamics Simulation ,Biochemistry ,Hydrophobic effect ,03 medical and health sciences ,Molecular dynamics ,Protein Domains ,Humans ,Amino Acid Sequence ,Molecular Biology ,Protein secondary structure ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,C-terminus ,Methanol ,Temperature ,Trifluoroethanol ,Electrostatics ,Protein Structure, Tertiary ,Intrinsically Disordered Proteins ,030104 developmental biology ,Polymorphism (materials science) ,CTD ,Tumor Suppressor Protein p53 ,Hydrophobic and Hydrophilic Interactions - Abstract
Proteins of the p53 family are best known for their role in the regulation of cell cycle. The p53 protein, as a model system, has been extensively explored in numerous cancer-related studies. The C-terminal domain (CTD) of p53 is an intrinsically disordered region that gains multiple different conformations at interaction with different binding partners. However, the impact of the surrounding environment on the structural preference of p53-CTD is not known. We investigated the impact of the surrounding environment on the conformational behavior and folding of p53-CTD. Although the entire CTD is predicted as a highly disordered region by several commonly used disorder predictors, based on the secondary structure prediction, we find that a part of the CTD sequence (residues 380-388) is "confused", being predicted to shuffle between the irregular, α-helical and β-strand structures. First time, we are observing the effect of folding-induced organic solvents, trifluoroethanol and methanol, on the conformation of CTD. Water-miscible organic solvents exert hydrophobic interactions, which are major driving force to trigger structural changes in CTD. By lowering the solution dielectric constant, organic solvents can also strengthen electrostatic interactions. We have also performed Replica Exchange Molecular Dynamic (REMD) simulations for enhanced conformation sampling of the peptide. These simulation studies have also provided detailed insight into the peculiarities of this peptide, explaining its folding behavior in the presence of methanol. We consider that these hydrophobic interactions may have important roles for function-related structural changes of this disordered region.
- Published
- 2020
20. Immunomodulatory and enhanced antitumor activity of a modified thymosin α1 in melanoma and lung cancer
- Author
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Fanwen Wang, Bin Li, Heng Zheng, Xingzhen Lao, Qingqing Li, and Pengcheng Fu
- Subjects
0301 basic medicine ,Lung Neoplasms ,Thymalfasin ,Recombinant Fusion Proteins ,T-Lymphocytes ,medicine.medical_treatment ,Mice, Nude ,Pharmaceutical Science ,Antineoplastic Agents ,Lymphocyte Activation ,Protein Structure, Secondary ,Mice ,03 medical and health sciences ,Adjuvants, Immunologic ,In vivo ,Cell Line, Tumor ,Immune Tolerance ,medicine ,Animals ,Humans ,Tumor-homing Peptide iRGD ,Lung cancer ,Melanoma ,Cell Proliferation ,CD86 ,Mice, Inbred BALB C ,Mice, Inbred ICR ,Chemistry ,Circular Dichroism ,Cancer ,Immunosuppression ,Trifluoroethanol ,medicine.disease ,Xenograft Model Antitumor Assays ,Fusion protein ,Mice, Inbred C57BL ,Thymosin ,030104 developmental biology ,Cancer research ,Female ,B7-2 Antigen ,Oligopeptides - Abstract
Tumor-targeted therapy is an attractive strategy for cancer treatment. Peptide hormone thymosin α1 (Tα1) has been used against several diseases, including cancer, but its activity is pleiotropic. Herein, we designed a fusion protein Tα1-iRGD by introducing the tumor homing peptide iRGD to Tα1. Results show that Tα1-iRGD can promote T-cell activation and CD86 expression, thereby exerting better effect and stronger inhibitory against melanoma and lung cancer, respectively, than Tα1 in vivo. These effects are indicated by the reduced densities of tumor vessels and Tα1-iRGD accumulation in tumors. Moreover, compared with Tα1, Tα1-iRGD can attach more B16F10 and H460 cells and exhibits significantly better immunomodulatory activity in immunosuppression models induced by hydrocortisone. Circular dichroism spectroscopy and structural analysis results revealed that Tα1 and Tα1-iRGD both adopted a helical confirmation in the presence of trifluoroethanol, indicating the structural basis of their functions. These findings highlight the vital function of Tα1-iRGD in tumor-targeted therapy and suggest that Tα1-iRGD is a better antitumor drug than Tα1.
- Published
- 2018
21. Inhibiting Human Calcitonin Fibril Formation with Its Most Relevant Aggregation-Resistant Analog
- Author
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Chian-Hui Lai, Bo Jie Huang, Yi Ting Chen, Ling Hsien Tu, and Kai Wei Hu
- Subjects
Calcitonin ,Protein Conformation, alpha-Helical ,Amyloid ,chemistry.chemical_element ,Peptide ,Calcium ,010402 general chemistry ,01 natural sciences ,Phosphates ,Protein Aggregates ,immune system diseases ,In vivo ,Salmon ,hemic and lymphatic diseases ,0103 physical sciences ,Bone cell ,Materials Chemistry ,Cyclic AMP ,Animals ,Humans ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Receptor ,Calcium metabolism ,chemistry.chemical_classification ,010304 chemical physics ,Trifluoroethanol ,0104 chemical sciences ,Surfaces, Coatings and Films ,Islet Amyloid Polypeptide ,surgical procedures, operative ,chemistry ,Mutation ,Cancer research ,MCF-7 Cells ,Sequence Alignment - Abstract
The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.
- Published
- 2019
22. Effect of trifluoroethanol on antibody reactivity against corresponding and nonrelated antigens
- Author
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S. V. Komisarenko, M. O. Demchenko, and S. A. Bobrovnik
- Subjects
0301 basic medicine ,Chemistry ,antibody affinity ,Biochemistry ,Molecular biology ,trifluoroethanol ,specific monoclonal antibodies ,lcsh:Biochemistry ,03 medical and health sciences ,030104 developmental biology ,Antigen ,antibody-antigen interaction ,polyreactive immunoglobulins ,lcsh:QD415-436 ,Antibody reactivity - Abstract
The ability of antibodies to switch between specific and nonspecific recognition of antigens under various factors is the key issue. Here we demonstrate that 2,2,2-trifluoroethanol (TFE) is one of these factors influencing the ability of monoclonal antibodies to react specifically with corresponding antigen (ovalbumin) and transforming them into polyreactive immunoglobulins (PRIGs) that are strong but nonspecific binders with various antigens. Such switching of antibody reactivity is nonlinear and even nonmonotonous function of TFE concentration and depends strongly on incubation time and temperature. At room temperatures (25 °C) the specific antibodies under 30% TFE action are transformed into PRIGs. However, at 0 °C the variation of antibody reactivity is complicated. TFE is known as the alcohol with one of the strongest proton-donor abilities in hydrogen bonding and its effect is probably in binding to specific sites that switch the antibody recognition ability.
- Published
- 2018
23. Temperature-Induced Phase Separation in Molecular Assembly of Nanotubes Comprising Amphiphilic Polypeptoid with Poly(N-ethyl glycine) in Water by a Hydrophilic-Region-Driven-Type Mechanism
- Author
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Tetsuya Hattori, Shunsaku Kimura, Toru Itagaki, and Hirotaka Uji
- Subjects
Sarcosine ,Metal Nanoparticles ,010402 general chemistry ,01 natural sciences ,Phase Transition ,chemistry.chemical_compound ,Phase (matter) ,Amphiphile ,Fluorescence Resonance Energy Transfer ,Materials Chemistry ,Physical and Theoretical Chemistry ,Nanotubes ,010405 organic chemistry ,Temperature ,Water ,Trifluoroethanol ,Temperature induced ,0104 chemical sciences ,Surfaces, Coatings and Films ,Förster resonance energy transfer ,chemistry ,Chemical engineering ,Colloidal gold ,Lateral diffusion ,Glycine ,Gold ,Peptides ,Hydrophobic and Hydrophilic Interactions - Abstract
Two kinds of amphiphilic polypeptoids having different types of hydrophilic polypeptoids, poly(sarcosine)-b-(l-Leu-Aib)6 (ML12) and poly(N-ethyl glycine)-b-(l-Leu-Aib)6 (EL12), were self-assembled via two paths to phase-separated nanotubes. One path was via sticking ML12 nanotubes with EL12 nanotubes and the other was a preparation from a mixture of ML12 and EL12 in solution. In either case, nanotubes showed temperature-induced phase separation along the long axis, which was observed by two methods of labeling one phase with gold nanoparticles and fluorescence resonance energy transfer between the components. The phase separation was ascribed to aggregation of poly(N-ethyl glycine) blocks over the cloud point temperature. The addition of 5% trifluoroethanol was needed for the phase separation because the tight association of the helices in the hydrophobic region should be loosened to allow lateral diffusion of the components to be separated. The phase separation in molecular assemblies in water based on t...
- Published
- 2018
24. 1,1,1,3,3,3-Hexafluoroisopropanol and 2,2,2-trifluoroethanol act more effectively on protein in combination than individually: Thermodynamic aspects
- Author
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Eva Judy, Nand Kishore, and Niki S. Jha
- Subjects
0301 basic medicine ,Circular dichroism ,ALPHA-LACTALBUMIN ,1,1,1,3,3,3-Hexafluoroisopropanol ,Enthalpy ,CYTOCHROME-C ,03 medical and health sciences ,chemistry.chemical_compound ,General Materials Science ,BOVINE SERUM-ALBUMIN ,Physical and Theoretical Chemistry ,Bovine serum albumin ,Protein secondary structure ,Thermal unfolding ,TRIFLUOROETHANOL ,Aqueous solution ,030102 biochemistry & molecular biology ,biology ,2,2,2-Trifluoroethanol ,Tryptophan ,Isothermal titration calorimetry ,MASS-SPECTROMETRY ,RIBONUCLEASE-A ,MOLTEN-GLOBULE STATE ,BETA-LACTOGLOBULIN ,Atomic and Molecular Physics, and Optics ,Crystallography ,030104 developmental biology ,chemistry ,FOLDING INTERMEDIATE ,biology.protein ,WATER MIXTURES - Abstract
2,2,2-Trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) are known to be secondary structure inducers of proteins. In order to determine the efficacy of TFE and HFIP in affecting the conformation of proteins when taken together, as compared to individually, we have studied the thermodynamics of unfolding of bovine serum albumin (BSA) in the presence of these alcohols along with the conformational characterization of the protein. A comparison of change in thermal transition temperature of the protein in the absence and presence of these alcohols in combination and individually shows that the (TFE+ HFIP) mixture is a stronger stabilizer of BSA up to a certain molality as compared to addition of their individual effects. The thermodynamics of effects of these alcohols on dithiotheitol reduced BSA were also studied. The enthalpies of interaction of TFE with HFIP in aqueous solution were measured by using isothermal titration calorimetry. The endothermic molar enthalpy of interaction of TFE with HFIP suggests that these alcohols do not strongly associate with each other through polar interactions. This is a possible explanation for their stronger effect on protein stability and conformation in combination rather than individually. The circular dichroism and fluorescence spectroscopic results provide evidence for the enhancement of the secondary structure of the protein by TFE and HFIP along with internalization of tryptophan residues in more hydrophobic environment. (C) 2018 Elsevier Ltd.
- Published
- 2018
25. Understanding the Role of Hydrophobic Terminal in the Hydrogen Bond Network of the Aqueous Mixture of 2,2,2-Trifluoroethanol: IR, Molecular Dynamics, Quantum Chemical as Well as Atoms in Molecules Studies
- Author
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Biswajit Biswas, Tonima Nandy, Saptarsi Mondal, and Prashant Chandra Singh
- Subjects
Spectrophotometry, Infrared ,Alcohol ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Chemical reaction ,chemistry.chemical_compound ,Molecular dynamics ,Computational chemistry ,0103 physical sciences ,Materials Chemistry ,Molecule ,Physical and Theoretical Chemistry ,Aqueous solution ,Ethanol ,010304 chemical physics ,Hydrogen bond ,Atoms in molecules ,Water ,Hydrogen Bonding ,Trifluoroethanol ,0104 chemical sciences ,Surfaces, Coatings and Films ,chemistry ,2,2,2-Trifluoroethanol ,Quantum Theory ,Hydrophobic and Hydrophilic Interactions - Abstract
The aqueous mixture of 2,2,2-trifluoroethanol (TFE) is one of the important alcoholic solvents which has been extensively used for understanding the stability of proteins as well as several chemical reactions. In this paper, the deconvolution of the IR lines in the OH-stretching region has been applied to understand the local structure of water–water, alcohol–water, and alcohol–alcohol interactions in the water mixture of TFE and ethanol (ETH). Further, molecular dynamics simulations, quantum chemical calculations, and atoms in molecules analysis have been performed to encode the local structure information obtained from the experimental data. Addition of a small amount of alcohol in a pure aqueous medium enhances the aggregation of water molecules for the case of ETH, whereas the hydrogen bond between TFE and water is the dominant contributor for TFE. The −CF3 substitution changes the orientation and hydrogen-bonding site of water molecules from the hydrophilic OH terminal to the hydrophobic −CF3 termina...
- Published
- 2018
26. SET-LRP of the Hydrophobic Biobased Menthyl Acrylate
- Author
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Joan Carlos Ronda, Marina Galià, Virgil Percec, Gerard Lligadas, Nabil Bensabeh, and Virginia Cádiz
- Subjects
Polymers and Plastics ,Radical polymerization ,Bioengineering ,Disproportionation ,02 engineering and technology ,Ligands ,010402 general chemistry ,01 natural sciences ,Catalysis ,Polymerization ,2-Propanol ,Electron Transport ,Biomaterials ,chemistry.chemical_compound ,Polymer chemistry ,Materials Chemistry ,Ethyl lactate ,Alkyl ,chemistry.chemical_classification ,Acrylate ,Ethanol ,Ligand ,Trifluoroethanol ,Bromine ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Acrylates ,chemistry ,Lactates ,Amine gas treating ,0210 nano-technology ,Hydrophobic and Hydrophilic Interactions ,Copper - Abstract
Cu(0) wire-catalyzed single electron transfer–living radical polymerization (SET-LRP) of (−)-menthyl acrylate, a biobased hydrophobic monomer, was investigated at 25 °C in ethanol, isopropanol, ethyl lactate, 2,2,2-trifluoroethanol (TFE), and 2,2,3,3-tetrafluoropropanol (TFP). All solvents are known to promote, in the presence of N ligands, the mechanistically required self-regulated disproportionation of Cu(I)Br into Cu(0) and Cu(II)Br2. Both fluorinated alcohols brought out their characteristics of universal SET-LRP solvents and showed the proper polarity balance to mediate an efficient polymerization of this bulky and hydrophobic monomer. Together with the secondary alkyl halide initiator, methyl 2-bromopropionate (MBP), and the tris(2-dimethylaminoethyl)amine (Me6-TREN) ligand, TFE and TPF mediated an efficient SET-LRP of MnA at room temperature that proceeds through a self-generated biphasic system. The results presented here demonstrate that Cu(0) wire-catalyzed SET-LRP can be used to target polyMnA...
- Published
- 2018
27. Can 2,2,2-trifluoroethanol be an efficient protein denaturant than methanol and ethanol under thermal stress?
- Author
-
Madhurima Jana and Dayanidhi Mohanta
- Subjects
Protein Denaturation ,Protein Folding ,Work (thermodynamics) ,Ethanol ,010304 chemical physics ,Hydrogen bond ,Methanol ,Temperature ,General Physics and Astronomy ,Alcohol ,Trifluoroethanol ,Molecular Dynamics Simulation ,010402 general chemistry ,Photochemistry ,01 natural sciences ,0104 chemical sciences ,Molecular dynamics ,chemistry.chemical_compound ,2,2,2-Trifluoroethanol ,chemistry ,0103 physical sciences ,Protein folding ,Physical and Theoretical Chemistry - Abstract
Monohydric alcohols, such as methanol (MEH), ethanol (ETH) and 2,2,2-trifluoroethanol (TFE), have significant effects on biological processes including the protein folding-unfolding phenomenon. Among the several monohydric alcohols, TFE, a fluorine-substituted alcohol, is known to induce a helical structure in proteins. In this work, we report the heterogeneous unfolding phenomenon of a small protein Chymotrypsin Inhibitor 2 in various concentrations of methanol, ethanol and TFE solutions by performing atomistic molecular dynamics simulation studies. Our study reveals that the unfolding phenomenon of CI2 under thermal stress majorly depends on the concentration and the nature of the alcohol. The presence of alcohols in general has been noted to accelerate the unfolding process compared to pure water and TFE, among them all, has been found to speed up the unfolding time scale at low concentrations. The molecular contact frequency between protein and alcohol follows the trend, MEHETHTFE at low concentrations, whereas the trend becomes MEH ∼ ETHTFE at more concentrated solutions. The differential water-mediated and self-clustering phenomena of alcohols, diverse protein-alcohol hydrogen bond strengths and the concentration dependent restricted inhomogeneous protein-water as well as protein-alcohol hydrogen bond dynamics suggest that TFE, a well known α-helix stabilizer, could be a good competitor among its class of denaturants.
- Published
- 2018
28. Extended disorder at the cell surface: The conformational landscape of the ectodomains of syndecans
- Author
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Frank Gondelaud, Fabien Chirot, Adam Liwo, Sylvie Ricard-Blum, Aurélien Le Fèvre, Bertrand Duclos, Adriana E. Miele, Mathilde Bouakil, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Spectrométrie des biomolécules et agrégats (SPECTROBIO), Institut Lumière Matière [Villeurbanne] (ILM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences Analytiques (ISA), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biophysics of complex systems, Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), ANABIO-MS - Analyse biomoléculaire par spectrométrie de masse - Biological Analysis by Mass Spectrometry, Catalyse Synthèse et Environnement (CASYEN), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), University of Gdańsk (UG), and Assemblages Supramoléculaires Péricellulaires et Extracellulaires (ASPE)
- Subjects
IDP ,GAG, glycosaminoglycan ,random-coil ,SDC ,Biochemistry ,TFE ,trifluoroethanol ,Syndecan 1 ,SAXS, small angle X-ray scattering ,ESI-MS, electrospray ionization - mass spectrometry ,Special Section on Molecular and Supramolecular structure of the extracellular matrix ,Edited by Sylvie Ricard-Blum ,solvent accessible surface area ,Biology (General) ,collision cross section ,CD, circular dichroism ,ectodomain ,SASA ,SDS ,TFE, trifluoroethanol ,DLS, dynamic light scattering ,GAG ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,biology ,Chemistry ,Extracellular matrix assembly ,cell-matrix interactions ,RC, random-coil ,dynamic light scattering ,size exclusion chromatography ,electrospray ionization - ion mobility - mass spectrometry ,SAXS ,SEC ,CSD, charge state distribution ,CD ,Ectodomain ,sodium dodecyl sulfate ,embryonic structures ,conformations ,ED, ectodomain ,DTT ,charge state distribution ,animal structures ,SDS, sodium dodecyl sulfate ,Syndecans ,Histology ,QH301-705.5 ,PMG, pre-molten globule ,DLS ,small angle X-ray scattering ,Integrin ,Biophysics ,MoRF, molecular recognition feature ,MoRF ,IDP, intrinsically disordered protein ,Intrinsically disordered proteins ,CSD ,Growth factor receptor ,SDC, syndecan ,ESI-IM-MS ,Cell surface receptor ,glycosaminoglycan ,Genetics ,ESI-IM-MS, electrospray ionization - ion mobility - mass spectrometry ,Cell adhesion ,electrospray ionization - mass spectrometry ,Molecular Biology ,PAGE, polyacrylamide gel electrophoresis ,CCS, collision cross section ,DTT, dithiothreitol ,SASA, solvent accessible surface area ,SEC, size exclusion chromatography ,molecular recognition feature ,PMG ,dithiothreitol ,ESI-MS ,Cell Biology ,intrinsically disordered protein ,ED ,CCS ,circular dichroism ,PAGE ,carbohydrates (lipids) ,biology.protein ,pre-molten globule ,polyacrylamide gel electrophoresis ,RC ,syndecan - Abstract
Highlights • The syndecan ectodomains are flexible random-coil intrinsically disordered proteins. • Their extended conformations have the same size as their cell surface partners. • Their binding-motifs to cells and receptors are not accessible in all conformations. • Only some of conformations of the ectodomains may be biologically active. • The syndecan-4 ectodomain forms a disulfide-bonded dimer with extended conformations., Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.
- Published
- 2021
29. Conformational behavior of alpha-2-macroglobulin: Aggregation and inhibition induced by TFE
- Author
-
Rizwan Hasan Khan, Mohammad Khalid Zia, Masihuz Zaman, Haseeb Ahsan, Ahmed Abdur Rehman, and Fahim Halim Khan
- Subjects
0301 basic medicine ,Circular dichroism ,Protein Conformation ,Protein aggregation ,Biochemistry ,Protein Aggregates ,03 medical and health sciences ,symbols.namesake ,Protein structure ,Structural Biology ,Animals ,Rayleigh scattering ,Molecular Biology ,Protein secondary structure ,Sheep ,Chemistry ,Trifluoroethanol ,General Medicine ,Pregnancy-Associated alpha 2-Macroglobulins ,Fluorescence ,Solvent ,Crystallography ,030104 developmental biology ,Solvents ,symbols ,Biophysics ,Protein folding ,Peptide Hydrolases - Abstract
Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body's innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α2M. TFE was sequentially added over a range of 0-20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α2M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α2M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α2M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α2M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α2M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α2M undergoes conformational and functional changes when exposed to TFE.
- Published
- 2017
30. Preparation and Synthetic Application of Naproxen-Containing Diaryliodonium Salts
- Author
-
Panpan Wu, Jun Zhou, Chao Chen, and Zhiyuan Bao
- Subjects
Naproxen ,Magnetic Resonance Spectroscopy ,Halogenation ,Chemistry, Pharmaceutical ,Pharmaceutical Science ,010402 general chemistry ,Ring (chemistry) ,01 natural sciences ,Catalysis ,Analytical Chemistry ,chemistry.chemical_compound ,Hydrolysis ,QD241-441 ,Phenols ,X-Ray Diffraction ,Trimethylsilyl trifluoromethanesulfonate ,Drug Discovery ,medicine ,naproxen methyl ester ,Organic chemistry ,Physical and Theoretical Chemistry ,Amination ,Dichloromethane ,Esterification ,010405 organic chemistry ,Communication ,Organic Chemistry ,Esters ,Stereoisomerism ,Fluorine ,Lipase ,Trifluoroethanol ,0104 chemical sciences ,Solvent ,chemistry ,diaryliodonium salts ,Chemistry (miscellaneous) ,Solvents ,Koser’s reagent ,functionalization ,Molecular Medicine ,Salts ,Iodine ,medicine.drug - Abstract
The synthesis of naproxen-containing diaryliodonium salts has been realized from naproxen methyl ester and ArI(OH)OTs activated by trimethylsilyl trifluoromethanesulfonate (TMSOTf) in a solvent mixture comprising dichloromethane and 2,2,2-trifluoroethanol (TFE). Those iodonium salts have been successfully used in the functionalization of an aromatic ring of naproxen methyl ester, including fluorination, iodination, alkynylation, arylation, thiophenolation, and amination and esterification reactions. Moreover, further hydrolysis of the obtained 5-iodo-naproxen methyl ester afforded 5-iodo-naproxen.
- Published
- 2021
31. Secondary structural alterations in glucoamylase as an influence of protein aggregation
- Author
-
Minhal Abidi, Afshin Iram, Aabgeena Naeem, and Mohammad Furkan
- Subjects
Protein Conformation, alpha-Helical ,0301 basic medicine ,Beta sheet ,Protein aggregation ,Ligands ,Biochemistry ,03 medical and health sciences ,Structural Biology ,Molecular Biology ,Protein secondary structure ,Dose-Response Relationship, Drug ,biology ,Chemistry ,Aspergillus niger ,Tryptophan ,Glyoxal ,Trifluoroethanol ,General Medicine ,biology.organism_classification ,Protein tertiary structure ,Molten globule ,Molecular Docking Simulation ,Kinetics ,Crystallography ,030104 developmental biology ,Protein Conformation, beta-Strand ,Glucan 1,4-alpha-Glucosidase ,Protein Multimerization ,Alpha helix - Abstract
Glucoamylase (EC 3.2.1.3) from Aspergillus niger possesses 31% α-helix, 36% β structure and rest aperiodic structure. A transition of glucoamylase structure in the presence of varying concentrations of glyoxal (GO) and trifluoroethanol (TFE) was studied by using multi-methodological approaches. At 20% GO, glucoamylase exists as molten globule state as evident by high tryptophan and ANS fluorescence, retention of secondary structure and loss of native tertiary structure. This state precedes the onset of the aggregation process and maximum is achieved at the highest concentration i.e. at 90% of GO. In parallel study TFE, on increasing concentration up to 25% induces secondary structure transformation leading to accumulation of intermolecular β sheets, altered tryptophan environment, high ANS and ThT fluorescence resulting in the formation of glucoamylase aggregates. Isothermal titration calorimetric curve is sigmoidal, indicating the weak binding of GO/TFE and glucoamylase. TEM studies showed that glucoamylase exists as globular and amorphous aggregates at 90% glyoxal and 25% TFE respectively. Further, TFE at 70% causes inhibition of enzyme aggregates; the majority of secondary structures observed at this concentration are α helices. Alpha helices being the main key player relocates glucoamylase native environment as evident by CD, FTIR and TEM. Hence induction of β sheet promotes protein aggregation and α helices inhibits protein aggregation.
- Published
- 2017
32. Enhanced cellular uptake and tumor penetration of nanoparticles by imprinting the 'hidden' part of membrane receptors for targeted drug delivery
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Chong Li, Na Li, Shan Peng, and Yahua Wang
- Subjects
Polymers ,Nanoparticle ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Catalysis ,Molecular Imprinting ,Mice ,Drug Delivery Systems ,Cell surface receptor ,Cell Line, Tumor ,Neoplasms ,Materials Chemistry ,Animals ,Humans ,Binding site ,Imprinting (psychology) ,Receptor ,Drug Carriers ,Binding Sites ,Chemistry ,fungi ,Metals and Alloys ,food and beverages ,Trifluoroethanol ,General Chemistry ,021001 nanoscience & nanotechnology ,Molecular biology ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Transmembrane domain ,Targeted drug delivery ,Drug delivery ,NIH 3T3 Cells ,Ceramics and Composites ,Biophysics ,Nanoparticles ,0210 nano-technology - Abstract
The widely existing transmembrane helices can serve as a novel type of binding site for recognizing corresponding membrane receptors. Through imprinting the transmembrane domain of certain receptors, here we report the construction of polymeric nanoparticles which can achieve enhanced cellular uptake and permeability in target tissues for tumor-targeted drug delivery.
- Published
- 2017
33. The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction
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Andrew B. Herr, Alexander E. Yarawsky, Lance R. English, and Steven T. Whitten
- Subjects
0301 basic medicine ,Proline ,Virulence Factors ,Glycine ,010402 general chemistry ,01 natural sciences ,Article ,Bacterial Adhesion ,Virulence factor ,Bacterial cell structure ,Methylamines ,03 medical and health sciences ,Bacterial Proteins ,Structural Biology ,Staphylococcus epidermidis ,Amino Acid Sequence ,Molecular Biology ,Polyproline helix ,Chemistry ,Biofilm ,Trifluoroethanol ,Adhesion ,0104 chemical sciences ,030104 developmental biology ,Biochemistry ,Osmolyte ,Biofilms ,Biophysics - Abstract
Staphylococcus epidermidis is one of the primary bacterial species responsible for healthcare-associated infections. The most significant virulence factor for S. epidermidis is its ability to form a biofilm, which renders the bacteria highly resistant to host immune responses and antibiotic action. Intercellular adhesion within the biofilm is mediated by the accumulation-associated protein (Aap), a cell wall-anchored protein that self-assembles in a zinc-dependent manner. The C-terminal portion of Aap contains a 135-aa-long, proline/glycine-rich region (PGR) that has not yet been characterized. The region contains a set of 18 nearly identical AEPGKP repeats. Analysis of the PGR using biophysical techniques demonstrated the region is a highly extended, intrinsically disordered polypeptide with unusually high polyproline type II helix propensity. In contrast to many intrinsically disordered polypeptides, there was a minimal temperature dependence of the global conformational state of PGR in solution as measured by analytical ultracentrifugation and dynamic light scattering. Furthermore, PGR was resistant to conformational collapse or α-helix formation upon the addition of the osmolyte trimethylamine N-oxide or the cosolvent 2,2,2-trifluoroethanol. Collectively, these results suggest PGR functions as a resilient, extended stalk that projects the rest of Aap outward from the bacterial cell wall, promoting intercellular adhesion between cells in the biofilm. This work sheds light on regions of low complexity often found near the attachment point of bacterial cell wall-anchored proteins.
- Published
- 2017
34. Amyloid-like aggregates formation by bovine apo-carbonic anhydrase in various alcohols: A comparative study
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Marjan Sabbaghian, Mohsen Nemat-Gorgani, Azadeh Ebrahim-Habibi, and Ali Es-haghi
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inorganic chemicals ,0301 basic medicine ,Amyloid ,Circular dichroism ,Time Factors ,Alcohol ,Protein aggregation ,Microscopy, Atomic Force ,Biochemistry ,Protein Aggregates ,03 medical and health sciences ,chemistry.chemical_compound ,Dynamic light scattering ,Structural Biology ,Carbonic anhydrase ,Metalloprotein ,Animals ,Benzothiazoles ,Molecular Biology ,Carbonic Anhydrases ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Trifluoroethanol ,General Medicine ,Fluorescence ,Dynamic Light Scattering ,Solutions ,Kinetics ,Thiazoles ,Crystallography ,030104 developmental biology ,Alcohols ,Hydrodynamics ,Biophysics ,biology.protein ,Cattle ,Hydrophobic and Hydrophilic Interactions - Abstract
Peptides and proteins convert from their native states to amyloid fibrillar aggregates in a number of pathological conditions. Characterizing these species could provide useful information on their pathogenicity and the key factors involved in their generation. In this study, we have observed the ability of the model protein apo-bovine carbonic anhydrase (apo-BCA) to form amyloid-like aggregates in the presence of halogenated and non-halogenated alcohols. Far-UV circular dichroism, ThT fluorescence, atomic force microscopy and dynamic light scattering were used to characterize these structures. The concentration required for effective protein aggregation varied between the solvents, with non-halogenated alcohols acting in a wider range. These aggregates show amyloid-like structures as determined by specific techniques used for characterizing amyloid structures. Oligomers were obtained with various size distributions, but fibrillar structures were not observed. Use of halogenated alcohols resulted into smaller hydrodynamic radii, and most stable oligomers were formed in hexafluoropropan-2-ol (HFIP). At optimal concentrations used to generate these structures, the non-halogenated alcohols showed higher hydrophobicity, which may be related to the lower stability of the generated oligomers. These oligomers have the potential to be used as models in the search for effective treatments in proteinopathies.
- Published
- 2016
35. About TFE: Old and New Findings
- Author
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Marilisa Leone, Marian Vincenzi, and Flavia Anna Mercurio
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Models, Molecular ,folding ,Protein Folding ,conformational transitions ,Molecular systems ,Intrinsically disordered proteins ,Biochemistry ,Phase Transition ,Protein Structure, Secondary ,TFE ,03 medical and health sciences ,alpha-helix ,Molecule ,Amino Acid Sequence ,Amino Acids ,amyloids ,Molecular Biology ,Protein secondary structure ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Chemistry ,030302 biochemistry & molecular biology ,Solvation ,Proteins ,Hydrogen Bonding ,secondary structure ,Trifluoroethanol ,Cell Biology ,General Medicine ,Protein tertiary structure ,Protein Structure, Tertiary ,IDPs ,Solvents ,Biophysics ,Thermodynamics ,Peptides ,Hydrophobic and Hydrophilic Interactions ,Protein Binding - Abstract
The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.
- Published
- 2019
36. Helical Structure of Recombinant Melittin
- Author
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Jayanti Pande, Alexander Shekhtman, and Lisa S. Ramirez
- Subjects
0301 basic medicine ,Glycerol ,Protein Conformation, alpha-Helical ,Stereochemistry ,Peptide ,Nuclear Overhauser effect ,010402 general chemistry ,01 natural sciences ,complex mixtures ,Melittin ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Tetramer ,Materials Chemistry ,Escherichia coli ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Carbon Isotopes ,Nitrogen Isotopes ,Chemistry ,Viscosity ,technology, industry, and agriculture ,Water ,Hydrogen Bonding ,Trifluoroethanol ,Melitten ,Recombinant Proteins ,0104 chemical sciences ,Surfaces, Coatings and Films ,030104 developmental biology ,Helix ,Protein folding ,lipids (amino acids, peptides, and proteins) - Abstract
Melittin is an extensively studied, 26-residue toxic peptide from honey bee venom. Because of its versatility in adopting a variety of secondary (helix or coil) and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies as well as in studies involving binding to proteins, lipids, and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of trifluoroethanol (TFE): a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using nuclear Overhauser effect (NOE)-derived distance restraints from three-dimensional NMR spectra, we determined the atomic resolution solution NMR structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFE-containing medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N- and C-terminal α-helices and the noncanonical 3(10)-helix in the middle. The noncanonical 3(10)-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin’s structure in TFE-containing medium provides insights into melittin’s conformational transitions, which are relevant to the peptide’s interactions with its biological targets.
- Published
- 2018
37. Effect of TFE on the Helical Content of AK17 and HAL-1 Peptides: Theoretical Insights into the Mechanism of Helix Stabilization
- Author
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Jiří Vymětal, Jiří Vondrášek, and Lucie Bednárová
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Protein Stability ,Chemistry ,Peptide ,Trifluoroethanol ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Surfaces, Coatings and Films ,03 medical and health sciences ,Molecular dynamics ,030104 developmental biology ,Protein stability ,Computational chemistry ,Materials Chemistry ,Biophysics ,Molecule ,Physical and Theoretical Chemistry ,Peptides ,Protein secondary structure - Abstract
Fluorinated alcohols such as 2,2,2-trifluoroethanol (TFE) are among the most frequently used cosolvents in experiment studies of peptides. They have significant effects on secondary structure and a particularly strong promotion of α-helix is induced by TFE. In this study we validated recently proposed force field parameters for TFE in molecular dynamics simulations with two model peptides-alanine-rich AK-17 and antimicrobial peptide halictine-1 (HAL-1). In the case of HAL-1, we characterized the effect of TFE on this peptide experimentally by ECD spectroscopy. Our TFE model in question reproduced the helix-promoting effect of TFE and provided insight into the mechanisms of TFE action on peptides. Our simulations confirmed the preferential interaction of TFE molecules with α-helices, although the TFE molecules accumulate in the vicinity of the peptides in various conformations. Moreover, we observed a significant effect of TFE on the thermodynamics of the helix-coil transition and a change in local conformational preferences in the unfolded (coil) state induced by TFE. In addition, our simulation-based analysis suggests that different mechanisms participate in helix stabilization in both model peptides in water and TFE solution. Our results thus support the picture of complex TFE action on peptides that is further diversified by the identity and intrinsic properties of the peptide.
- Published
- 2016
38. Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE
- Author
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Ricardo Barros Mariutti, F.R. De Morais, D. Rehders, Ícaro Putinhon Caruso, Raghuvir K. Arni, Anwar Ullah, Universidade Estadual Paulista (Unesp), and Hamburg University
- Subjects
0301 basic medicine ,Viral genome-linked protein ,Circular dichroism ,Protein Conformation ,Molecular Sequence Data ,Gene Expression ,Expression ,Plasma protein binding ,Biology ,Viral Nonstructural Proteins ,Biochemistry ,Plant Viruses ,03 medical and health sciences ,Protein structure ,Dynamic light scattering ,Structural Biology ,Nucleotide ,Histidine ,Amino Acid Sequence ,Peptide sequence ,Molecular Biology ,Purification ,chemistry.chemical_classification ,VPg ,Nucleotides ,RNA ,Fabaceae ,General Medicine ,Trifluoroethanol ,030104 developmental biology ,chemistry ,Protein Binding - Abstract
Made available in DSpace on 2018-12-11T16:59:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-01 Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes. Multiuser Center for Biomolecular Innovation IBILCE/UNESP Department of Physics IBILCE/UNESP Laboratory for Structural Biology of Infection and Inflammation Hamburg University Multiuser Center for Biomolecular Innovation IBILCE/UNESP Department of Physics IBILCE/UNESP
- Published
- 2016
- Full Text
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39. An Interesting Class of Porous Polymer-Revisiting the Structure of Mesoporous α-<scp>d</scp>-Polysaccharide Gels
- Author
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Peter S. Shuttleworth, Vitaliy L. Budarin, James H. Clark, Anna Fischer, Robin J. White, and Mario De bruyn
- Subjects
General Chemical Engineering ,Stereoisomerism ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Diffusion ,chemistry.chemical_compound ,Polysaccharides ,Amylose ,Phase (matter) ,Environmental Chemistry ,Organic chemistry ,Molecule ,General Materials Science ,chemistry.chemical_classification ,Chemistry ,Sorption ,Trifluoroethanol ,Polymer ,Carbon-13 NMR ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,General Energy ,Chemical engineering ,0210 nano-technology ,Mesoporous material ,Gels ,Porosity - Abstract
The processes involved in the transformation of non-porous, native polysaccharides to their highly porous equivalents introduce significant molecular complexity and are not yet fully understood. In this paper, we propose that distinct changes in polysaccharide local short-range ordering promotes and directs the formation of meso- and micro-pores, which are investigated here using N2 sorption, FTIR, and solid-state (13)C NMR. It is found that an increase in the overall double helical amylose content, and their local association structures, are responsible for formation of the porous polysaccharide gel phase. An exciting consequence of this local ordering change is elegantly revealed using a (19)F NMR experiment, which identifies the stereochemistry-dependent diffusion of a fluorinated chiral probe molecule (1-phenyl-2,2,2-trifluoroethanol) from the meso- to the micro-pore region. This finding opens opportunities in the area of polysaccharide-based chiral stationary phases and asymmetric catalyst preparation.
- Published
- 2016
40. Solution Structure of Enterocin HF, an Antilisterial Bacteriocin Produced by Enterococcus faecium M3K31
- Author
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Pablo E. Hernández, John C. Vederas, Sara Arbulu, Marco J. van Belkum, Luis M. Cintas, Christopher T. Lohans, and Carmen Herranz
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Circular dichroism ,Magnetic Resonance Spectroscopy ,biology ,Protein Conformation ,Stereochemistry ,Circular Dichroism ,Enterococcus faecium ,Trifluoroethanol ,General Chemistry ,Nuclear Overhauser effect ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,Antiparallel (biochemistry) ,Amino acid ,Solutions ,Protein structure ,Bacteriocins ,chemistry ,Biochemistry ,Bacteriocin ,General Agricultural and Biological Sciences - Abstract
The solution structure of enterocin HF (EntHF), a class IIa bacteriocin of 43 amino acids produced by Enterococcus faecium M3K31, was evaluated by CD and NMR spectroscopy. Purified EntHF was unstructured in water, but CD analysis supports that EntHF adopts an α-helical conformation when exposed to increasing concentrations of trifluoroethanol. Furthermore, NMR spectroscopy indicates that this bacteriocin adopts an antiparallel β-sheet structure in the N-terminal region (residues 1-17), followed by a well-defined central α-helix (residues 19-30) and a more disordered C-terminal end (residues 31-43). EntHF could be structurally organized into three flexible regions that might act in a coordinated manner. This is in agreement with the absence of long-range nuclear Overhauser effect signals between the β-sheet domain and the C-terminal end of the bacteriocin. The 3D structure recorded for EntHF fits emerging facts regarding target recognition and mode of action of class IIa bacteriocins.
- Published
- 2015
41. Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells
- Author
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Victor Carramiñana, Ana M. Ochoa de Retana, Francisco Palacios, and Jesús M. de los Santos
- Subjects
Allylic rearrangement ,Double bond ,Phosphines ,Phosphorous Acids ,Ethanethiol ,Organophosphonates ,Pharmaceutical Science ,Adenocarcinoma of Lung ,Antineoplastic Agents ,010402 general chemistry ,01 natural sciences ,Medicinal chemistry ,Article ,Analytical Chemistry ,lcsh:QD241-441 ,chemistry.chemical_compound ,α-aminophosphine oxide or phosphonate acetals ,Phenols ,lcsh:Organic chemistry ,Nucleophile ,Drug Discovery ,Humans ,Sulfhydryl Compounds ,Physical and Theoretical Chemistry ,2H-azirines ,chemistry.chemical_classification ,Nucleophilic addition ,Azirines ,010405 organic chemistry ,allylic α-aminophosphorus compounds ,Organic Chemistry ,Regioselectivity ,Stereoisomerism ,Trifluoroethanol ,Aziridine ,Phosphonate ,0104 chemical sciences ,Oxygen ,antiproliferative effect ,chemistry ,aziridines ,Chemistry (miscellaneous) ,Molecular Medicine ,Drug Screening Assays, Antitumor ,Sulfur - Abstract
This work reports a straightforward regioselective synthetic methodology to prepare &alpha, aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C&ndash, N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different &alpha, aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford &alpha, aminophosphine oxide and phosphonate acetals after N&ndash, C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic &alpha, aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some &alpha, aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.
- Published
- 2020
42. Structural properties and enzyme stabilization function of the intrinsically disordered LEA_4 protein TdLEA3 from wheat
- Author
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Dirk K. Hincha, Sana Koubaa, Anne Bremer, and Faiçal Brini
- Subjects
0301 basic medicine ,Models, Molecular ,Circular dichroism ,Amino Acid Motifs ,lcsh:Medicine ,Intrinsically disordered proteins ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Lactate dehydrogenase ,Enzyme Stability ,Spectroscopy, Fourier Transform Infrared ,Glycerol ,Molecule ,lcsh:Science ,Triticum ,Plant Proteins ,chemistry.chemical_classification ,Multidisciplinary ,Sequence Homology, Amino Acid ,Circular Dichroism ,lcsh:R ,Trifluoroethanol ,Amino acid ,Intrinsically Disordered Proteins ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,lcsh:Q ,030217 neurology & neurosurgery - Abstract
Late Embryogenesis Abundant (LEA) proteins are mostly predicted to be intrinsically disordered proteins (IDPs) that are induced under conditions of cellular dehydration. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been investigated in a small number of proteins. Here, we have characterized the wheat LEA protein TdLEA3, which has sequence homology with the group of LEA_4 proteins that are characterized by the 11-mer repeat motif TAQAAKEKAXE. TdLEA3 has five repeats of this imperfectly conserved 11-mer amino acid motif. To investigate the structure of the protein, we used circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. The data show that TdLEA3 was largely disordered under fully hydrated conditions and acquired α-helical structure upon drying and in the presence of trifluoroethanol (TFE). Moreover, the addition of increasing glycerol concentrations to the protein solution induced a progressive gain in α-helix content. Activity assays indicated that TdLEA3 was able to prevent the inactivation of lactate dehydrogenase (LDH) under heat, dehydration-rehydration and freeze-thaw treatments. In addition, TdLEA3 reduced aggregate formation in the enzyme during these treatments.
- Published
- 2018
43. Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution
- Author
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Gregory F. Pirrone, Nicole M. Schiavone, Erik D. Guetschow, Alexey A. Makarov, and Ian Mangion
- Subjects
Protein Conformation, alpha-Helical ,Circular dichroism ,Protein Folding ,Size-exclusion chromatography ,Peptide ,02 engineering and technology ,Mass spectrometry ,01 natural sciences ,Mass Spectrometry ,Analytical Chemistry ,Humans ,Thermal stability ,Protein secondary structure ,chemistry.chemical_classification ,Chemistry ,Circular Dichroism ,010401 analytical chemistry ,Deuterium Exchange Measurement ,Trifluoroethanol ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Solutions ,Biophysics ,Chromatography, Gel ,Protein folding ,Protein Conformation, beta-Strand ,0210 nano-technology ,Peptides ,Higher Order Structure - Abstract
Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and β-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques – circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.
- Published
- 2018
44. Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies
- Author
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Douglas Duarte Lopes, Luciana Malavolta, Jamille H Cuvero, Rogério Lauria da Silva, Clovis R. Nakaie, Shirley Schreier, Sinval E. G. Souza, and Mariana Mendonça Ferreira
- Subjects
0301 basic medicine ,Circular dichroism ,Protein Conformation ,Clinical Biochemistry ,Peptide ,Biochemistry ,Receptors, G-Protein-Coupled ,Cyclic N-Oxides ,03 medical and health sciences ,chemistry.chemical_compound ,Peptide synthesis ,Amino Acid Sequence ,Spin label ,Conformational isomerism ,Chromatography, High Pressure Liquid ,Solid-Phase Synthesis Techniques ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Circular Dichroism ,Organic Chemistry ,Electron Spin Resonance Spectroscopy ,Trifluoroethanol ,Transmembrane protein ,Microspheres ,Peptide Fragments ,Transmembrane domain ,Crystallography ,030104 developmental biology ,chemistry ,Solvents ,Spin Labels ,Hydrophobic and Hydrophilic Interactions ,Macromolecule - Abstract
We compared the synthesis and structural/conformational details of the (66-97) segments of the second transmembrane helix of AT1, MAS and B2, all of which belong to the class of G-protein-coupled receptors (GPCR). Step-by-step monitoring of the coupling reactions during the growth of these transmembrane peptides revealed that the increase in the level of difficulty started at the 6-10 regions of the sequence. Possibly due to their long and hydrophobic sequences, the final estimated synthesis yields decreased progressively by up to 20-25%. Analytical high pressure liquid chromatography showed that the hydrophobicity indexes of each TM-8, -16, -24 and -32 segments correlated linearly with their retention time. Microscopic measurements of peptide-resin beads indicated that, in general, dichloromethane and dimethylsulfoxide were the best solvents for solvating resin beads in the initial and final stages of the synthesis, respectively. Results from electron paramagnetic resonance experiments with Toac (2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled peptide resins revealed that the level of peptide chain mobility throughout the polymer network was in agreement with their swelling data measured in different solvents. Initial results regarding conformational features determined by circular dichroism (CD) spectra revealed typical α-helicoidally structures for MAS and B2 TM32 fragments when in more than roughly 30% (v/v) trifluoroethanol (TFE). In contrast, the AT1-TM32 segment revealed CD spectra, more representatives of a mixture of other secondary helical conformers, regardless of the amount of TFE. These findings observed in different aspects of these receptors' fragments support further investigations of GPCR-type macromolecules.
- Published
- 2018
45. A novel fluorinated triazole derivative suppresses macrophage activation and alleviates experimental colitis via a Twist1-dependent pathway
- Author
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Zhen Huang, Junqing Hu, Jianlin Han, Lei Dong, Wei He, Yi Pan, Junfeng Zhang, Jingjing Gan, Mao Yu, Yanping Zhang, Xiafei Shi, Jinhao Xu, and Tingyue Tu
- Subjects
0301 basic medicine ,Male ,Repressor ,Biochemistry ,Small hairpin RNA ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Animals ,Colitis ,Acute colitis ,Cells, Cultured ,Pharmacology ,Mice, Knockout ,Mice, Inbred BALB C ,Mice, Inbred ICR ,Microarray analysis techniques ,Chemistry ,Twist-Related Protein 1 ,Nuclear Proteins ,Biological activity ,Trifluoroethanol ,Macrophage Activation ,Triazoles ,medicine.disease ,Mice, Inbred C57BL ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer research ,Tumor necrosis factor alpha ,Female ,Signal transduction ,Signal Transduction - Abstract
Hyperactivated macrophages play a key role in the initiation and perpetuation of mucosal inflammation in Crohn's disease (CD). Increasing evidence suggests that the basic helix-loop-helix (bHLH) repressor Twist1 can suppress activation of nuclear factor-κB (NF-κB) and the subsequent production of TNF-α, which are both essential elements of macrophage activation. Thus, developing novel therapeutic strategies to enhance Twist1 expression and to inhibit macrophage activation may be beneficial for CD treatment. In the present study, a series of trifluoroethyl thiazolo[3,2-b][1,2,4]triazole derivatives were used to investigate their potential anti-inflammatory activities and the underlying mechanism. In a biological activity screen, compound 7# (Thiazolo[3,2-b][1,2,4]triazole-5-methanamine, 6-phenyl-α-(trifluoromethyl)-, (αR)-, TT-TFM) suppressed the activation of macrophages. Consistent with the in vitro data, TT-TFM protected against 2,4,6-trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS)-induced acute colitis and IL-10 knockout (KO) chronic colitis, as judged by body weight changes and colonic pathological damage. A mechanistic study based on microarray analysis and gene interference experiments indicated that TT-TFM exerted anti-inflammatory effects by enhancing Twist1 expression and subsequently blocking the NF-κB/TNF-α pathway. In addition, pretreatment with lentiviruses encoding shRNA targeting Twist1 could abolish the therapeutic effect of TT-TFM in TNBS colitis. Ultimately, TT-TFM showed anti-colitis activity by reducing NF-κB activation and the TNF-α level by promoting Twist1 expression; thus, TT-TFM may offer a therapeutic strategy for CD patients.
- Published
- 2018
46. A biophysical insight into the formation of aggregates upon trifluoroethanol induced structural and conformational changes in garlic cystatin
- Author
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Bilqees Bano and Mohd Faizan Siddiqui
- Subjects
0301 basic medicine ,Circular dichroism ,Protein Folding ,Protein aggregation ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Aggregates ,Garlic ,Instrumentation ,Protein secondary structure ,Spectroscopy ,Plant Proteins ,Circular Dichroism ,food and beverages ,Trifluoroethanol ,Cystatins ,Atomic and Molecular Physics, and Optics ,Molten globule ,Congo red ,030104 developmental biology ,chemistry ,Plant protein ,Biophysics ,Protein folding ,Cystatin - Abstract
Intrinsic and extrinsic factors are responsible for the transition of soluble proteins into aggregated form. Trifluoroethanol is among such potent extrinsic factor which facilitates the formation of aggregated structure. It disrupts the interactive forces and destabilizes the native structure of the protein. The present study investigates the effect of trifluoroethanol (TFE) on garlic cystatin. Garlic cystatin was incubated with increasing concentration of TFE (0-90% v/v) for 4 h. Incubation of GPC with TFE induces structural changes thereby resulting in the formation of aggregates. Inactivation of garlic phytocystatin was confirmed by cysteine proteinase inhibitory activity. Garlic cystatin at 30% TFE exhibits native-like secondary structure and high ANS fluorescence, thus suggesting the presence of molten globule state. Circular dichroism and FTIR confirmed the transition of the native alpha-helical structure of garlic cystatin to the beta-sheet structure at 60% TFE. Furthermore, increased ThT fluorescence and redshift in Congo red absorbance assay confirmed the presence of aggregates. Rayleigh and turbidity assay was also performed to validate the aggregation results. Scanning electron microscopy was followed to analyze the morphological changes which confirm the presence of sheath-like structure at 60% TFE. The study sheds light on the conformational behavior of a plant protein when kept under stress condition induced by an extrinsic factor.
- Published
- 2018
47. Alterations in local stability and dynamics of A4V SOD1 in the presence of trifluoroethanol
- Author
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Andrew M. Lynn, Vijay Kumar, and Amresh Prakash
- Subjects
0301 basic medicine ,Circular dichroism ,SOD1 ,Biophysics ,Protein aggregation ,Molecular Dynamics Simulation ,Biochemistry ,Protein Structure, Secondary ,Biomaterials ,03 medical and health sciences ,Molecular dynamics ,Superoxide Dismutase-1 ,Catalytic Domain ,Enzyme Stability ,Humans ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Protein dynamics ,Circular Dichroism ,Organic Chemistry ,Amyotrophic Lateral Sclerosis ,nutritional and metabolic diseases ,Energy landscape ,Active site ,General Medicine ,Trifluoroethanol ,Kinetics ,030104 developmental biology ,Mutation ,biology.protein ,Protein folding ,Spectrophotometry, Ultraviolet ,Hydrophobic and Hydrophilic Interactions - Abstract
Alterations in the local dynamics of Cu/Zn Superoxide dismutase (SOD1) due to mutations affect the protein folding, stability, and function leading to misfolding and aggregation seen in amyotrophic lateral sclerosis (ALS). Here, we study the structure and dynamics of the most devastating ALS mutation, A4V SOD1 in aqueous trifluoroethanol (TFE) through experiments and simulation. Far-UV circular dichroism (CD) studies shows that TFE at intermediate concentrations (∼15% - 30%) induce partially unfolded β-sheet-rich extended conformations in A4V SOD1 which subsequently aggregates. Molecular dynamics (MD) simulation results shows that A4V SOD1 increases local dynamics in the active site loops that leads to the destabilization of the β-barrel and loss of hydrophobic contacts, thus stipulating a basis for aggregation. Free energy landscape (FEL) and essential dynamics (ED) analysis demonstrates the conformational heterogeneity in A4V SOD1. Our results thus shed light on the role of local unfolding and conformational dynamics in aggregation of SOD1.
- Published
- 2017
48. Fluoroalcohol - Induced coacervates for selective enrichment and extraction of hydrophobic proteins
- Author
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Morteza G. Khaledi, Halie Rion, Armin Oloumi, Ariel O'Brien, Amir Koolivand, and Stephan Clayton
- Subjects
Proteomics ,Propanols ,Clinical Biochemistry ,Liquid-Liquid Extraction ,02 engineering and technology ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Pulmonary surfactant ,Phase (matter) ,Amphiphile ,Chromatography, High Pressure Liquid ,Chromatography, Reverse-Phase ,Aqueous solution ,Chromatography ,Coacervate ,Cetrimonium ,010401 analytical chemistry ,Membrane Proteins ,Cell Biology ,General Medicine ,Trifluoroethanol ,Hydrogen-Ion Concentration ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Partition coefficient ,chemistry ,Chemical engineering ,Ionic strength ,Gramicidin ,Cetrimonium Compounds ,0210 nano-technology ,Hydrophobic and Hydrophilic Interactions - Abstract
As previously reported, fluoroalcohols can induce coacervation in aqueous solutions of amphiphilic compounds with subsequent formation of two-phase systems, where one phase is enriched in amphiphile and fluoroalcohol and the other is primarily an aqueous – rich phase. This study focuses on the use of simple coacervates made of a single component amphiphile induced by a fluoroalcohol for extraction and enrichment of proteins. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) were used to induce coacervation in the aqueous solutions of a cationic amphiphile, cetyltrimethylammonium bromide (CTAB) or tetra-n-butylammonium bromide (TBAB). Cationic amphiphiles (CTAB, TBAB) formed two-phase coacervate systems in a basic pH and/or sufficient ionic strength depending on the strength of coacervator (HFIP or TFE). The phase diagrams for TBAB paired with HFIP or TFE coacervates were created. By increasing the concentration of coacervator (HFIP or TFE) at a constant surfactant concentration, transition from a single liquid phase to a two or multiple phase mixture, and then eventually to a single liquid phase was observed. TBAB/HFIP mixture without additives showed a unique three-phase system before transitioning to a two-phase system upon increasing HFIP concentration. However, salt addition eliminated this three-phase region and expanded the region of two-phase formation. Select two-phase systems composed of TBAB and a perfluoroalcohol (HFIP or TFE) were utilized to extract model proteins of ranging hydrophobicity. All coacervate phases extracted bacteriorhodopsin, a membrane protein, and gramicidin, a very hydrophobic polypeptide ion channel. The most hydrophilic protein in the mixture, ribonuclease A, remained in aqueous phases. The coacervates formed from TBAB/TFE/200 mM NaCl mixture and TBAB/HFIP mixture exhibited the most selectivity in extracting proteins of high hydrophobicity. The partition coefficient (P) for each protein was calculated using the ratio of the protein concentration in the coacervate to that in the aqueous-rich phases. TBAB (50 mM)/HFIP (8%, v/v) coacervate showed remarkable selectivity and a high partition coefficient (>100) for both bacteriorhodopsin and gramicidin. Thus, this system may potentially be beneficial for facile fractionation of hydrophobic and membrane proteins in proteomics applications.
- Published
- 2017
49. Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix
- Author
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Yuji Goto, Tsuyoshi Konuma, Masanori Yagi, Satoshi Takahashi, Kazumasa Sakurai, and Tetsuro Fujisawa
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Folding ,Circular dichroism ,Protein Conformation ,Chemistry ,Small-angle X-ray scattering ,Circular Dichroism ,Lactoglobulins ,Trifluoroethanol ,Protein Structure, Secondary ,Folding (chemistry) ,Crystallography ,Protein structure ,X-Ray Diffraction ,Structural Biology ,Scattering, Small Angle ,Helix ,Radius of gyration ,Animals ,Cattle ,Protein folding ,Molecular Biology ,Protein secondary structure - Abstract
In the folding of β-lactoglobulin (βLG), a predominantly β-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of βLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200μs. Time-resolved SAXS showed that the radius of gyration (R(g)) of the intermediate at 300 μs was 23.3±0.7 Å, indicating a considerable collapse from the unfolded state having R(g) of 35.1±7.1 Å. Further compaction to R(g) of 21.2±0.3 Å occurred with a time constant of 28±11 ms. Pair distribution functions showed that the intermediate at 300 μs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar R(g) of 23.0±0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of βLG.
- Published
- 2015
50. Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes
- Author
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Akira Shigenaga, Keiichiro Okuhira, Fuka Ogata, Hiroyuki Saito, Akira Otaka, Kohei Tsuji, Toshinori Shimanouchi, Chiharu Mizuguchi, Shiho Mikawa, and Teruhiko Baba
- Subjects
Amyloid ,Recombinant Fusion Proteins ,Mutant ,Gene Expression ,Peptide ,macromolecular substances ,Protein Engineering ,Fibril ,Biochemistry ,Protein Structure, Secondary ,Structure-Activity Relationship ,chemistry.chemical_compound ,polycyclic compounds ,Escherichia coli ,Humans ,Benzothiazoles ,Lipid bilayer ,Molecular Biology ,Unilamellar Liposomes ,Fluorescent Dyes ,chemistry.chemical_classification ,Apolipoprotein A-I ,Chemistry ,nutritional and metabolic diseases ,Trifluoroethanol ,Cell Biology ,Random coil ,Protein Structure, Tertiary ,Amino acid ,Thiazoles ,Mutation ,Protein Structure and Folding ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Thioflavin ,Protein Binding - Abstract
The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.
- Published
- 2015
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