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  • 1
    Language: English
    In: Accounts of chemical research, 2018-09-18, Vol.51 (9), p.1911-1920
    Description: Conspectus Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors’ active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature’s nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme’s diffusion due to active self-propulsion must be observed on top of the enzyme’s passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme’s substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme’s diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes’ catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.
    ISSN: 0001-4842
    E-ISSN: 1520-4898
    Source: Hellenic Academic Libraries Link
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  • 2
    Language: English
    In: The Journal of chemical physics, 2019-03-28, Vol.150 (12), p.124201-124201
    Description: The diffusion of enzymes is of fundamental importance for many biochemical processes. Enhanced or directed enzyme diffusion can alter the accessibility of substrates and the organization of enzymes within cells. Several studies based on fluorescence correlation spectroscopy report enhanced diffusion of enzymes upon interaction with their substrate or inhibitor. In this context, major importance is given to the enzyme fructose-bisphosphate aldolase, for which enhanced diffusion has been reported even though the catalysed reaction is endothermic. Additionally, enhanced diffusion of tracer particles surrounding the active aldolase enzymes has been reported. These studies suggest that active enzymes can act as chemical motors that self-propel and give rise to enhanced diffusion. However, fluorescence studies of enzymes can, despite several advantages, suffer from artefacts. Here, we show that the absolute diffusion coefficients of active enzyme solutions can be determined with Pulsed Field Gradient Nuclear Magnetic Resonance (PFG-NMR). The advantage of PFG-NMR is that the motion of the molecule of interest is directly observed in its native state without the need for any labelling. Furthermore, PFG-NMR is model-free and thus yields absolute diffusion constants. Our PFG-NMR experiments of solutions containing active fructose-bisphosphate aldolase from rabbit muscle do not show any diffusion enhancement for the active enzymes, nor the surrounding molecules. Additionally, we do not observe any diffusion enhancement of aldolase in the presence of its inhibitor pyrophosphate.
    Subject(s): Rabbits ; Animals ; Fructose-Bisphosphate Aldolase - chemistry ; Magnetic Resonance Spectroscopy - methods ; Diffusion
    ISSN: 0021-9606
    E-ISSN: 1089-7690
    Source: American Institute of Physics (AIP) Publications
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  • 3
    Language: English
    In: Nature communications, 2020-09-09, Vol.11 (1), p.4513-4513
    Description: Abstract Chiral plasmonic nanoparticles can exhibit strong chiroptical signals compared to the corresponding molecular response. Observations are, however, generally restricted to measurements on stationary single particles with a fixed orientation, which complicates the spectral analysis. Here, we report the spectroscopic observation of a freely diffusing single chiral nanoparticle in solution. By acquiring time-resolved circular differential scattering signals we show that the spectral interpretation is significantly simplified. We experimentally demonstrate the equivalence between time-averaged chiral spectra observed for an individual nanostructure and the corresponding ensemble spectra, and thereby demonstrate the ergodic principle for chiroptical spectroscopy. We also show how it is possible for an achiral particle to yield an instantaneous chiroptical response, whereas the time-averaged signals are an unequivocal measure of chirality. Time-resolved chiroptical spectroscopy on a freely moving chiral nanoparticle advances the field of single-particle spectroscopy, and is a means to obtain the true signature of the nanoparticle’s chirality.
    Subject(s): Nanoparticles ; Nanophotonics and plasmonics ; Circular dichroism
    ISSN: 2041-1723
    E-ISSN: 2041-1723
    Source: Nature Open Access
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 4
    Language: English
    In: BMC plant biology, 2018-10-22, Vol.18 (1), p.251-12
    Description: Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic β-cyanoalanine may accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles. A BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa. While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2 and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced in Escherichia coli accepted biogenic nitriles such as β-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed nitrile hydratase activity towards β-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic parameters of PtNIT1 suggest that the enzyme utilizes β-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed, β-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced β-cyanoalanine accumulation is likely caused by ethylene formation. Our data suggest a role for PtNIT1 in the catabolism of herbivore-induced β-cyanoalanine and benzyl cyanide in poplar leaves.
    Subject(s): Biological research ; Enzymes ; Physiological aspects ; Poplar ; Nitriles ; Hydrolases ; Genetic aspects ; Research ; Plant-animal interactions ; Biology, Experimental ; Analysis ; Escherichia coli ; Ethylene ; Asparagine ; Explosions ; Benzyl cyanide ; Populus trichocarpa ; NIT1 ; Plant defense ; β-Cyanoalanine ; Nitrilase
    ISSN: 1471-2229
    E-ISSN: 1471-2229
    Source: BioMedCentral Open Access
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 5
    Language: English
    In: The journal of physical chemistry letters, 2021-07-01, Vol.12 (25), p.5932-5937
    Description: A study reported in The Journal of Physical Chemistry Letters (Wang et al., 2021, 12, 2370) of “boosted mobility” measured by diffusion NMR experiments contains significant errors in data analysis and interpretation. We carefully reanalyzed the same data and find no evidence of boosted mobility, and we identify several sources of error.
    ISSN: 1948-7185
    E-ISSN: 1948-7185
    Source: Hellenic Academic Libraries Link
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  • 6
    Language: English
    In: Plant physiology (Bethesda), 2019-06-01, Vol.180 (2), p.767-782
    Description: Upon herbivory, the tree species western balsam poplar (Populus trichocarpa) produces a variety of Phe-derived metabolites, including 2-phenylethylamine, 2-phenylethanol, and 2-phenylethyl-beta-D-glucopyranoside. To investigate the formation of these potential defense compounds, we functionally characterized aromatic L-amino acid decarboxylases (AADCs) and aromatic aldehyde synthases (AASs), which play important roles in the biosynthesis of specialized aromatic metabolites in other plants. Heterologous expression in Escherichia coli and Nicotiana benthamiana showed that all five AADC/AAS genes identified in the P. trichocarpa genome encode active enzymes. However, only two genes, PtAADC1 and PtAAS1, were significantly upregulated after leaf herbivory. Despite a sequence similarity of similar to 96%, PtAADC1 and PtAAS1 showed different enzymatic functions and converted Phe into 2-phenylethylamine and 2-phenylacetaldehyde, respectively. The activities of both enzymes were interconvertible by switching a single amino acid residue in their active sites. A survey of putative AADC/AAS gene pairs in the genomes of other plants suggests an independent evolution of this function-determining residue in different plant families. RNA interference-mediated-downregulation of AADC1 in gray poplar (Populus x canescens) resulted in decreased accumulation of 2-phenylethylamine and 2-phenylethyl-beta-D-glucopyranoside, whereas the emission of 2-phenylethanol was not influenced. To investigate the last step of 2-phenylethanol formation, we identified and characterized two P. trichocarpa short-chain dehydrogenases, PtPAR1 and PtPAR2, which were able to reduce 2-phenylacetaldehyde to 2-phenylethanol in vitro. In summary, 2-phenylethanol and its glucoside may be formed in multiple ways in poplar. Our data indicate that PtAADC1 controls the herbivore-induced formation of 2-phenylethylamine and 2-phenylethyl-beta-D-glucopyranoside in planta, whereas PtAAS1 likely contributes to the herbivore-induced emission of 2-phenylethanol.
    Subject(s): Life Sciences & Biomedicine ; Plant Sciences ; Science & Technology ; Poplar ; Physiological aspects
    ISSN: 0032-0889
    E-ISSN: 1532-2548
    Source: American Society of Plant Biologists
    Source: Hellenic Academic Libraries Link
    Source: Web of Science - Science Citation Index Expanded - 2019〈img src="http://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /〉
    Source: PubMed Central
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  • 7
    Language: English
    In: ACS nano, 2019-05-28, Vol.13 (5), p.5810-5815
    Description: Catalytically active colloids are model systems for chemical motors and active matter. It is desirable to replace the inorganic catalysts and the toxic fuels that are often used with biocompatible enzymatic reactions. However, compared to inorganic catalysts, enzyme-coated colloids tend to exhibit less activity. Here, we show that the self-assembly of genetically engineered M13 bacteriophages that bind enzymes to magnetic beads ensures high and localized enzymatic activity. These phage-decorated colloids provide a proteinaceous environment for directed enzyme immobilization. The magnetic properties of the colloidal carrier particle permit repeated enzyme recovery from a reaction solution, while the enzymatic activity is retained. Moreover, localizing the phage-based construct with a magnetic field in a microcontainer allows the enzyme–phage–colloids to function as an enzymatic micropump, where the enzymatic reaction generates a fluid flow. This system shows the fastest fluid flow reported to date by a biocompatible enzymatic micropump. In addition, it is functional in complex media including blood, where the enzyme-driven micropump can be powered at the physiological blood-urea concentrations.
    Subject(s): Physical Sciences ; Chemistry ; Materials Science ; Nanoscience & Nanotechnology ; Technology ; Materials Science, Multidisciplinary ; Science & Technology - Other Topics ; Chemistry, Multidisciplinary ; Chemistry, Physical ; Science & Technology
    ISSN: 1936-0851
    E-ISSN: 1936-086X
    Source: Hellenic Academic Libraries Link
    Source: Web of Science - Science Citation Index Expanded - 2019〈img src="http://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /〉
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  • 8
    Language: English
    In: ACS nano, 2019-10-22, Vol.13 (10), p.11453-11459
    Description: Plasmonic molecules are building blocks of metallic nanostructures that give rise to intriguing optical phenomena with similarities to those seen in molecular systems. The ability to design plasmonic hybrid structures and molecules with nanometric resolution would enable applications in optical metamaterials and sensing that presently cannot be demonstrated, because of a lack of suitable fabrication methods allowing the structural control of the plasmonic atoms on a large scale. Here we demonstrate a wafer-scale “lithography-free” parallel fabrication scheme to realize nanogap plasmonic meta-molecules with precise control over their size, shape, material, and orientation. We demonstrate how we can tune the corresponding coupled resonances through the entire visible spectrum. Our fabrication method, based on glancing angle physical vapor deposition with gradient shadowing, permits critical parameters to be varied across the wafer and thus is ideally suited to screen potential structures. We obtain billions of aligned dimer structures with controlled variation of the spectral properties across the wafer. We spectroscopically map the plasmonic resonances of gold dimer structures and show that they not only are in good agreement with numerically modeled spectra, but also remain functional, at least for a year, in ambient conditions.
    Subject(s): Physical Sciences ; Chemistry ; Materials Science ; Nanoscience & Nanotechnology ; Technology ; Materials Science, Multidisciplinary ; Science & Technology - Other Topics ; Chemistry, Multidisciplinary ; Chemistry, Physical ; Science & Technology
    ISSN: 1936-0851
    E-ISSN: 1936-086X
    Source: Hellenic Academic Libraries Link
    Source: Web of Science - Science Citation Index Expanded - 2019〈img src="http://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /〉
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  • 9
    Language: English
    In: Plant physiology (Bethesda), 2019-06, Vol.180 (2), p.767-782
    Description: Biochemical analysis of the aromatic amino acid decarboxylase family in poplar revealed two enzymes involved in the herbivore induced formation of 2-phenylethanol and 2-phenylethylamine. Upon herbivory, the tree species western balsam poplar ( Populus trichocarpa ) produces a variety of Phe-derived metabolites, including 2-phenylethylamine, 2-phenylethanol, and 2-phenylethyl-β- d -glucopyranoside. To investigate the formation of these potential defense compounds, we functionally characterized aromatic l -amino acid decarboxylases (AADCs) and aromatic aldehyde synthases (AASs), which play important roles in the biosynthesis of specialized aromatic metabolites in other plants. Heterologous expression in Escherichia coli and Nicotiana benthamiana showed that all five AADC / AAS genes identified in the P . trichocarpa genome encode active enzymes. However, only two genes, PtAADC1 and PtAAS1 , were significantly upregulated after leaf herbivory. Despite a sequence similarity of ∼96%, PtAADC1 and PtAAS1 showed different enzymatic functions and converted Phe into 2-phenylethylamine and 2-phenylacetaldehyde, respectively. The activities of both enzymes were interconvertible by switching a single amino acid residue in their active sites. A survey of putative AADC / AAS gene pairs in the genomes of other plants suggests an independent evolution of this function-determining residue in different plant families. RNA interference -mediated–downregulation of AADC1 in gray poplar ( Populus × canescens ) resulted in decreased accumulation of 2-phenylethylamine and 2-phenylethyl-β- d -glucopyranoside, whereas the emission of 2-phenylethanol was not influenced. To investigate the last step of 2-phenylethanol formation, we identified and characterized two P . trichocarpa short-chain dehydrogenases, PtPAR1 and PtPAR2, which were able to reduce 2-phenylacetaldehyde to 2-phenylethanol in vitro. In summary, 2-phenylethanol and its glucoside may be formed in multiple ways in poplar. Our data indicate that PtAADC1 controls the herbivore-induced formation of 2-phenylethylamine and 2-phenylethyl-β- d -glucopyranoside in planta, whereas PtAAS1 likely contributes to the herbivore-induced emission of 2-phenylethanol.
    ISSN: 0032-0889
    E-ISSN: 1532-2548
    Source: American Society of Plant Biologists
    Source: Hellenic Academic Libraries Link
    Source: PubMed Central
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  • 10
    Language: English
    In: Plant signaling & behavior, 2019-11-02, Vol.14 (11), p.1668233-1668233
    Description: In response to herbivory, poplar produces among other compounds the volatile alcohol 2-phenylethanol and its corresponding glucoside 2-phenylethyl-β-D-glucopyranoside. While the free alcohol is released only upon herbivory, the glucoside accumulates also in undamaged leaves, but increases after herbivore feeding. Recently we showed that 2-phenylethanol and its glucoside are biosynthesized via separate pathways in Populus trichocarpa. The phenylacetaldehyde synthase PtAAS1 plays a central role in the de novo formation of herbivory-induced volatile 2-phenylethanol, while the phenylalanine decarboxylase PtAADC1 initiates a pathway responsible for the herbivory-induced production of 2-phenylethyl-β-D-glucopyranoside. Besides PtAAS1, P. trichocarpa possesses another aromatic aldehyde synthase PtAAS2 with in vitro enzymatic activity comparable to that of PtAAS1. However, in contrast to PtAAS1, which is exclusively expressed in herbivory-damaged leaves, PtAAS2 was found to be expressed at constant levels in both damaged and undamaged leaves. Thus it has been hypothesized that PtAAS2 provides phenylacetaldehyde as substrate for the constitutive formation of 2-phenylethyl-β-D-glucopyranoside in undamaged P. trichocarpa trees. By generating RNAi-mediated AAS2 knockdown plants, we show here that despite the similar activities of PtAAS1 and PtAAS2 in vitro, the latter enzyme does not contribute to the biosynthesis of 2-phenylethyl-β-D-glucopyranoside in planta. Based on the recent finding that phenylpyruvic acid accumulates in undamaged poplar leaves, the constitutive formation of the glucoside may now be suggested to proceed via the Ehrlich pathway, which begins with the conversion of phenylalanine into phenylpyruvic acid.
    Subject(s): 2-phenylethanol ; herbivory ; 2-phenylethyl-β-D-glucopyranoside ; Populus trichocarpa ; aldehyde synthase ; Biochemistry & Molecular Biology ; Life Sciences & Biomedicine ; Plant Sciences ; Science & Technology
    ISSN: 1559-2316
    ISSN: 1559-2324
    E-ISSN: 1559-2324
    Source: Taylor & Francis Open Access
    Source: Web of Science - Science Citation Index Expanded - 2019〈img src="http://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /〉
    Source: PubMed Central
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