Single molecular assay of individual ATP turnover by a myosin‐GFP fusion protein expressed in vitro

Iwane, Atsuko H.; Funatsu, Takashi; Harada, Yoshie; Tokunaga, Makio; Ohara, Osamu; Morimoto, Sachiko; Yanagida, Toshio

doi:10.1016/s0014-5793(97)00359-1

Cited by 53 publications

(43 citation statements)

References 20 publications

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“…Applications of single-molecule spectroscopy to biomolecules have included studies of conformational fluctuations of individual DNA molecules [233], hydrolysis of individual ATP molecules by a single molecule of myosin [204,235], myosin moving along an actin rod [236], individual kinesin molecules moving along microtubules [235,237,238], interconversions of the bound flavin in cholesterol oxidase between its oxidized and reduced states [239], and elucidation of pathways for folding of proteins and ribozymes [234,[240][241][242][243][244][245][246][247][248]. A powerful extension of the technique is to measure resonance energy transfer between a pair of fluorescent dyes attached to a macromolecule.…”

Section: Single-molecule Fluorescence and High-resolution Fluorescencmentioning

confidence: 99%

Fluorescence

Parson

2015

Modern Optical Spectroscopy

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Section: Single-molecule Fluorescence and High-resolution Fluorescencmentioning

confidence: 99%

Fluorescence

Parson

2015

Modern Optical Spectroscopy

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“…For this purpose imaging onto multichannel detectors [28][29][30][31][32][33][34], confocal microscopy [31][32][35][36], and near field scanning optical microscopy (NSOM) [37,38] were used (Fig. 2).…”

Section: Imaging Experimentsmentioning

confidence: 99%

“…The very first successful detection of single GFP molecules for in vitro experiments was reported by Iwane et al for an elegant colocalization study involving a myosin-GFP chimera and fluorescently labeled ATP [28]. In these experiments the myosin fragment S1dC was fused with the Integration times were 5 ms with a time lag of 16 ms between each frame.…”

Section: Single Molecule Detection With Gfp As a Labelmentioning

confidence: 99%

Single Molecule Spectroscopy of the Green Fluorescent Protein: A Critical Assessment

Zumbusch

Jung

2000

Single Mol.

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“…These species are available for applications in multicolor labeling and fluorescence energy transfer. Moreover, recent progress in fluorescence microscopy has enabled us to visualize individual GFP molecules (8,12).We applied these advantages of GFP to the detection of bacteria. To express GFP within bacteria, a bacteriophage containing the GFP gene (GFP phage) was Microbiol.…”

mentioning

confidence: 99%

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confidence: 99%

Rapid and Sensitive Detection Method of a Bacterium by Using a GFP Reporter Phage

Funatsu

Taniyama

Tajima

et al. 2002

Microbiology and Immunology

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The fields of medicine and public health require the rapid detection of bacterial pathogens that cause severe infectious diseases and poisoning. The conventional method for detecting a bacterial pathogen makes use of the antibodies against it. This method has some drawbacks: Its sensitivity is insufficient; bacteria must be purified from biological samples; and the process takes many hours. Polymerase chain reaction (PCR) has recently been used to detect bacteria and viruses (4,5,13). This method provides improved sensitivity, but it too is laborious as well as expensive. Therefore a rapid, convenient, and sensitive method remains eagerly awaited. Here we present a new detection method that uses green fluorescent protein (GFP) (14). GFP from the jellyfish Aequorea victoria is a very useful tool as a reporter in molecular and cellular biology (2, 18). GFP absorbs UV light at 395 nm and emits fluorescence with an emission peak at 510 nm. The chromophore is generated by spontaneous cyclization and oxidation of a serine-dehydrotyrosine-glycine trimer within a defined hexapeptide sequence of the protein (3). Unlike the luciferase reporter gene (9), for example, exogenous substrates and cofactors are not required for GFP fluorescence. Recently, variants of the GFP and GFP-like fluorescence proteins, such as red fluorescent proteins, have been isolated (17). These species are available for applications in multicolor labeling and fluorescence energy transfer. Moreover, recent progress in fluorescence microscopy has enabled us to visualize individual GFP molecules (8,12).We applied these advantages of GFP to the detection of bacteria. To express GFP within bacteria, a bacteriophage containing the GFP gene (GFP phage) was Microbiol. Immunol., 46(6), 365 369, 2002 365 Abbreviations: GFP, green fluorescent protein; GFP phage, λTriplEx phage carrying the gene of green fluorescent protein; IPTG, isopropyl-β-D-thiogalactopyranoside; LB medium, a solution containing 1% NaCl, 1% Bacto-tryptone, 0.5% Bacto-yeast extract, pH 7.5; PCR, polymerase chain reaction; TBS-T, a solution containing 10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween-20. Abstract: A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4 6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection o...

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Single molecular assay of individual ATP turnover by a myosin‐GFP fusion protein expressed in vitro

Cited by 53 publications

References 20 publications

Fluorescence

Fluorescence

Single Molecule Spectroscopy of the Green Fluorescent Protein: A Critical Assessment

Rapid and Sensitive Detection Method of a Bacterium by Using a GFP Reporter Phage

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