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  1. Article ; Online: Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

    Poddar, Souvik / Levitus, Marcia

    The journal of physical chemistry. B

    2024  Volume 128, Issue 11, Page(s) 2640–2651

    Abstract: 2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA- ... ...

    Abstract 2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H
    MeSH term(s) 2-Aminopurine/chemistry ; Fluorescence ; DNA/chemistry ; Carbonates ; Phosphates ; Spectrometry, Fluorescence/methods
    Chemical Substances 2-Aminopurine (452-06-2) ; DNA (9007-49-2) ; Carbonates ; Phosphates
    Language English
    Publishing date 2024-03-07
    Publishing country United States
    Document type Journal Article
    ISSN 1520-5207
    ISSN (online) 1520-5207
    DOI 10.1021/acs.jpcb.3c07269
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples.

    Levitus, Marcia

    Methods and applications in fluorescence

    2020  Volume 8, Issue 3, Page(s) 33001

    Abstract: The measurement of fluorescence spectra and the determination of fluorescence quantum yields in transparent samples are conceptually simple tasks, but these procedures are subject to several pitfalls that can lead to significant errors. Available ... ...

    Abstract The measurement of fluorescence spectra and the determination of fluorescence quantum yields in transparent samples are conceptually simple tasks, but these procedures are subject to several pitfalls that can lead to significant errors. Available technical reports and protocols often assume that the reader possesses a solid theoretical background in spectroscopy and has ample experience with fluorescence instrumentation, but this is often not the case given the many applications of fluorescence in diverse fields of science. The goal of this tutorial is to provide a didactic treatment of the topic that will hopefully be accessible to readers without extensive expertise in the field of fluorescence. The article covers the theoretical background needed to understand the origins of the most common artifacts researchers can expect. Possible artifacts are illustrated with examples to help readers avoid them or identify them if present. A step-by-step example of a fluorescence quantum yield determination in solution is provided with detailed experimental information to help readers understand how to design and analyze experiments.
    Language English
    Publishing date 2020-04-22
    Publishing country England
    Document type Journal Article
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/ab7e10
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  3. Article ; Online: Otto Wolfbeis 1947-2023.

    Birch, David J S / Levitus, Marcia / Mély, Yves

    Methods and applications in fluorescence

    2023  Volume 12, Issue 1

    Language English
    Publishing date 2023-12-13
    Publishing country England
    Document type Editorial
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/ad12f8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Quantity with quality.

    Birch, David J S / Levitus, Marcia / Mély, Yves

    Methods and applications in fluorescence

    2023  Volume 11, Issue 1

    Language English
    Publishing date 2023-01-03
    Publishing country England
    Document type Journal Article
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/aca5f6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: MAF

    Birch, David J S / Levitus, Marcia / Mély, Yves

    Methods and applications in fluorescence

    2021  Volume 10, Issue 1

    Language English
    Publishing date 2021-12-14
    Publishing country England
    Document type Editorial
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/ac3ec0
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: MAF and fluorescence play their part.

    Birch, David J S / Levitus, Marcia / Mély, Yves

    Methods and applications in fluorescence

    2020  Volume 9, Issue 1

    MeSH term(s) Fluorescence
    Language English
    Publishing date 2020-12-23
    Publishing country England
    Document type Editorial
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/abce06
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Relaxation Kinetics by Fluorescence Correlation Spectroscopy: Determination of Kinetic Parameters in the Presence of Fluorescent Impurities.

    Levitus, Marcia

    The journal of physical chemistry letters

    2010  Volume 1, Page(s) 1346–1350

    Abstract: The use of Fluorescence Correlation Spectroscopy (FCS) in combination with Förster Resonance Energy Transfer (FRET) is gaining popularity as a tool to investigate kinetics in equilibrium conditions. The technique is based on the study of fluorescence ... ...

    Abstract The use of Fluorescence Correlation Spectroscopy (FCS) in combination with Förster Resonance Energy Transfer (FRET) is gaining popularity as a tool to investigate kinetics in equilibrium conditions. The technique is based on the study of fluorescence fluctuations in small numbers of molecules, and is particularly well-suited to investigate conformational dynamics in biopolymers. In practice, its applicability is often hindered by the presence of certain impurities such as partially labeled biomolecules, excess of free fluorophore, or partially dissociated multi-subunit complexes. Here, we show that the simultaneous measurement of the fluctuations in the donor and acceptor intensities allows the determination of the kinetic relaxation time of the reaction in the presence of donor-only particles when cross-talk is negligible, or in cases where all species have the same diffusion coefficient. Theoretical predictions are supported with the results of Monte Carlo simulations, and demonstrate that the applicability of the technique is more general than previously thought.
    Language English
    Publishing date 2010-03-28
    Publishing country United States
    Document type Journal Article
    ISSN 1948-7185
    ISSN (online) 1948-7185
    DOI 10.1021/jz100231v
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Photophysical properties of the hemicyanine Dy-630 and its potential as a single-molecule fluorescent probe for biophysical applications.

    Kumari, Nikita / Ciuba, Monika A / Levitus, Marcia

    Methods and applications in fluorescence

    2019  Volume 8, Issue 1, Page(s) 15004

    Abstract: Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction ... ...

    Abstract Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2-4 -fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.
    MeSH term(s) Benzopyrans/chemistry ; Biophysics ; DNA/chemistry ; DNA/metabolism ; Fluorescence ; Fluorescent Dyes/chemistry ; Indoles/chemistry ; Molecular Structure ; Photochemical Processes ; Proteins/chemistry ; Proteins/metabolism ; Viscosity
    Chemical Substances Benzopyrans ; DY 630 ; Fluorescent Dyes ; Indoles ; Proteins ; DNA (9007-49-2)
    Language English
    Publishing date 2019-11-12
    Publishing country England
    Document type Journal Article
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/ab4b0d
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Potassium Glutamate and Glycine Betaine Induce Self-Assembly of the PCNA and β-Sliding Clamps.

    Purohit, Anirban / Douma, Lauren G / Bloom, Linda B / Levitus, Marcia

    Biophysical journal

    2020  Volume 120, Issue 1, Page(s) 73–85

    Abstract: Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the β-clamp is of the ... ...

    Abstract Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the β-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of the β-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the Saccharomyces cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the β-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.
    MeSH term(s) Betaine ; DNA Replication ; Escherichia coli/metabolism ; Escherichia coli Proteins ; Glutamic Acid ; Proliferating Cell Nuclear Antigen/metabolism
    Chemical Substances Escherichia coli Proteins ; Proliferating Cell Nuclear Antigen ; Glutamic Acid (3KX376GY7L) ; Betaine (3SCV180C9W)
    Language English
    Publishing date 2020-11-19
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 218078-9
    ISSN 1542-0086 ; 0006-3495
    ISSN (online) 1542-0086
    ISSN 0006-3495
    DOI 10.1016/j.bpj.2020.11.013
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Photophysical characterization of interchromophoric interactions between rhodamine dyes conjugated to proteins.

    Donaphon, Bryan / Bloom, Linda B / Levitus, Marcia

    Methods and applications in fluorescence

    2018  Volume 6, Issue 4, Page(s) 45004

    Abstract: Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of ... ...

    Abstract Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E. coli sliding clamp (β clamp) with emphasis on the properties of the H-dimers that form when the dimeric protein is labeled with one dye at each side of the dimer interface. Overall, results are consistent with an equilibrium between non-emissive dimers and unstacked monomers that experience efficient dynamic quenching Protein constructs labeled with tetramethylrhodamine show the characteristic features of H-dimers in their absorption spectra and a c.a. 40-fold quenching of fluorescence intensity. The degree of quenching decreases when samples are labeled with a tetramethylrhodamine derivative bearing a six-carbon linker. H-dimers do not form in samples labeled with Alexa 488 and A546, but fluorescence is still quenched in these samples through a dynamic mechanism. These results should help researchers design and interpret fluorescence experiments that take advantage of the properties of rhodamine dimers in protein research.
    MeSH term(s) Humans ; Proteins/metabolism ; Rhodamines/metabolism ; Spectrometry, Fluorescence/methods
    Chemical Substances Proteins ; Rhodamines
    Language English
    Publishing date 2018-07-26
    Publishing country England
    Document type Journal Article
    ISSN 2050-6120
    ISSN (online) 2050-6120
    DOI 10.1088/2050-6120/aad20f
    Database MEDical Literature Analysis and Retrieval System OnLINE

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