Direct Observation of Collective Blinking and Energy Transfer in a Bichromophoric System

Tinnefeld, Philip; Buschmann, Volker; Weston, Kenneth D.; Sauer, Markus

doi:10.1021/jp026565u

Cited by 60 publications

(119 citation statements)

References 36 publications

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“…Similar traces were observed with the active L93C NiR (no ascorbate/PES, no nitrite) and with glass slides spin-coated with an ATTO 665 solution in 1% polyvinyl alcohol. Periods of high fluorescence alternate on a time scale of seconds with dark periods in which only the background signal is observed; we ascribe this to dye blinking (21)(22)(23)(24). The steady nature of the observed fluorescence during the ''on'' periods is consistent with crystallographic and NMR evidence, indicating absence of largescale conformational fluctuations (18,25).…”

Section: Resultssupporting

confidence: 71%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A generic method is described for the fluorescence ''readout'' of the activity of single redox enzyme molecules based on Fö rster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to singlemolecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.electron transfer ͉ redox enzyme ͉ Fö rster transfer ͉ nitric oxide ͉ fluorescent label I n the past few years, single-enzyme studies have revealed numerous hidden aspects of enzyme behavior (1). The huge potential of these studies to unravel the intricate kinetics and precise workings of enzymes, which are often hidden within the ensemble properties, is somewhat restricted by current approaches. The majority of existing single-molecule enzymatic assays are based on fluorescence and have been limited to the flavoenzymes, which contain a fluorescent cofactor (2-5), or to enzymes for which a suitable fluorogenic substrate could be designed (6-9). Recently, it was shown how redox enzyme activity in the bulk can be studied by Förster resonance energy transfer (FRET) from an attached fluorescent label to the enzyme cofactor (10, 11). Here, we report, first, how this technique can be successfully applied to study the enzymatic turnover of single surface-confined copper-containing nitrite reductase (NiR) molecules labeled with ATTO 655 using scanning confocal fluorescence microscopy. Second, it is shown how the kinetics of the fluorescence time traces can be connected with the kinetic parameters that describe the ensemble behavior of the enzyme. Finally, the rate of the electron transfer between the types 1 and 2 Cu centers during turnover could be established. The observed distribution of rates is connected to the partial structural disorder in the catalytic site that has been observed in crystallographic studies. ResultsEnzyme Mechanism. Dissimilatory copper-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6 converts nitrite into nitric oxide. The enzyme is a homotrimer, each monomer containing one type 1 and one type 2 copper site (Fig. 1). The type 1 copper accepts an electron from the physiological donor and transfers it to the type 2 copper, where nitrite is reduced to nitric oxide (NO). The midpoint potentials of the types 1 and 2 Cu centers are close, resulting in a redox equilibrium constant for the two sites that is close to 1 (12, 13). Regarding the enzyme mechanism, it has been stated (14, 15) that, after reduction of the type 1 Cu site, the electron is passed on to the type 2...

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Section: Resultssupporting

confidence: 71%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, in vacuum, triplet state lifetimes of 4-100 ms have been measured with a triplet yield of 0.04% for single DiIC12 molecules (a carbocyanine derivative) [32]. Similar values were measured for fluorophores immobilized in thin polymer films such as PVA with low oxygen permeability [33,34]. Alternatively, one can remove oxygen from aqueous solutions using efficient enzymatic scavenger systems as used in the STORM and dSTORM methods [7,8,11,15,35,36].…”

Section: Resultsmentioning

confidence: 65%

Photoswitching microscopy with standard fluorophores

et al. 2008

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We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires widefield illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

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“…For example, in vacuum, triplet state lifetimes of 4-100 ms have been measured with a triplet yield of 0.04% for single DiIC12 molecules (a carbocyanine derivative) [173]. Similar values were measured for fluorophores immobilized in thin polymer films such as PVA with low oxygen permeability [174,175]. Alternatively, one can remove oxygen from aqueous solutions using efficient enzymatic scavenger systems as used in the STORM and dSTORM method [59][60][61][62]64,176,177].…”

Section: Discussionmentioning

confidence: 86%

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Heilemann

Dedecker

Hofkens

et al. 2009

Laser & Photonics Reviews

248

191

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Optical microscopes, often referred to as 'light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluorophores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation "Photoswitching Microscopy". Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subcellular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.All subdiffraction-resolution fluorescence imaging methods are based on the separation of fluorescence emission in time. Thus, they critically depend on the availability of photoswitches, i.e. molecules that can be switched between a fluorescent and nonfluorescent state upon irradiation with light with high reliability. Here we describe the development and operation methods of diverse photoswitches and their application for superresolution fluorescence imaging and molecular quantification.

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Direct Observation of Collective Blinking and Energy Transfer in a Bichromophoric System

Cited by 60 publications

References 36 publications

The enzyme mechanism of nitrite reductase studied at single-molecule level

The enzyme mechanism of nitrite reductase studied at single-molecule level

Photoswitching microscopy with standard fluorophores

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

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