Energy Transfer. A System with Relatively Fixed Donor-Acceptor Separation

Latt, Samuel A.; Cheung, H. T. Andrew; Blout, E. R.

doi:10.1021/ja01083a011

Cited by 195 publications

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“…Electronic excitation energy can be efficiently transferred between a fluorescent energy donor (such as fluorescein) and a suitable energy acceptor (such as eosin) over distances of the order of 50 A. This transfer process depends on the inverse sixth power of the distance between the donor and acceptor (11,18,29). Hence, we expected that a donor-labeled actin would transfer energy to an adjacent acceptor-labeled actin but not to a more distant one.…”

mentioning

confidence: 99%

Detection of actin assembly by fluorescence energy transfer.

Taylor¹,

Reidler²,

Spudich³

et al. 1981

The Journal of Cell Biology

128

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A fundamental problem in cell motility is to define the state of assembly and interaction of the major cytoskeletal and contractile proteins both in vitro and in vivo . One example is the role played by actin assembly and disassembly during motile events . To date, actin assembly has usually been assayed indirectly both in vitro and in vivo. Experiments using viscosity, light scattering, and electron microscopy yield results that could be interpreted in several ways (2,10,12,13,20,44; see Taylor and Condeelis [32] and Uyemura and Spudich [37] for reviews) . Furthermore, more direct measurements of assembly using sedimentation assays (3,25) can only be applied in vitro. Therefore, techniques must be developed that would yield direct evidence for the state of actin assembly and interaction with specific molecules even in living cells.Fluorescence spectroscopy has already generated some information in vitro on the assembly of actin (4, 6, 40), the structure of actin (14,15,19,21,31), and the interaction of actin with associated proteins (6,23,24).I Recently, the technique of molecular cytochemistry (33,40; see Taylor and Wang [39] for a review) has opened the possibility of making spectroscopic measurements of fluorescently labeled actin in living cells. Actin labeled with 5-iodoacetamidofluorescein has been studied initially, because the site of labeling is known, the extinction coefficient is high, and it is excited by a relatively long wavelength of light (495 nm), which minimizes autofluorescence and cellular damage (40) .Dr. P. Detmers kindly provided D. L. Taylor with a copy of her manuscript (6) before publication. ABSTRACTFluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide) . Donor-labeled actin and acceptor-labeled actin were coassembled . The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is -35 A, which means that this site is located near the outer surface of the filament . The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.Fluorescence energy transfer (7, 28) is a powerful technique for studying the structure and dynamics of molecular...

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mentioning

confidence: 99%

Detection of actin assembly by fluorescence energy transfer.

Taylor¹,

Reidler²,

Spudich³

et al. 1981

The Journal of Cell Biology

128

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“…Intermolecular transfer of electronic excitation energy, which depends on the inverse sixth power of the separation between donor and acceptor dyes (1), has been extensively used to examine proximity relationships in well-defined biochemical systems (2)(3)(4)(5) Values for R0, the critical distance for 50% efficient energy transfer between different dye pairs, were calculated from the * To whom reprint requests should be sent. …”

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confidence: 99%

“…Intermolecular transfer of electronic excitation energy, which depends on the inverse sixth power of the separation between donor and acceptor dyes (1), has been extensively used to examine proximity relationships in well-defined biochemical systems (2)(3)(4)(5). Energy transfer between dyes in more complex biological structures can also in principle be used to detect systematic differences between donor and acceptor binding domains.…”

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confidence: 99%

Enhancement of banding patterns in human metaphase chromosomes by energy transfer

Sahar¹,

Latt²

1978

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

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Intermolecular energy transfer between appropriately chosen pairs of dyes can be used to induce or enhance banding patterns in human metaphase chromosomes. Intermolecular transfer of electronic excitation energy, which depends on the inverse sixth power of the separation between donor and acceptor dyes (1), has been extensively used to examine proximity relationships in well-defined biochemical systems (2)(3)(4)(5) Values for R0, the critical distance for 50% efficient energy transfer between different dye pairs, were calculated from the * To whom reprint requests should be sent. 5650The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…13 Forster has proposed that the transfer occurs by a dipole-dipole resonance interaction between the energy donor and acceptor chromophores. In his theory, the rate constant for transfer kT is related to geometric and spectroscopic factors by5 kT = r-JK2 n-4 kF X 8.71 X 1023 sec-1 (1) where r is the distance (in A) between the centers of the donor and acceptor transition moments, K2 is the dipole-dipole orientation factor, n is the refractive index of the medium, and kF is the rate constant (in sec1) for fluorescence emission by the donor. The spectral overlap integral J, which measures the extent to which the donor and acceptor transitions are in resonance, is given by = fF(X)e(X)dX (2) where F (X) is the fluorescence intensity of the energy donor at wavelength X (in cm), and E(X) is the molar decadic extinction coefficient (in cm-' M-) of the energy acceptor.…”

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confidence: 99%

Dependence of the Kinetics of Singlet-Singlet Energy Transfer on Spectral Overlap

Haugland

Yguerabide

Stryer

1969

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

162

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Abstract.-Electronic excitation energy can be transferred between chromophores separated by distances of the order of 30 A. Forster proposed that the transfer occurs by a dipole-dipole resonance interaction which depends on certain spectroscopic and geometric properties of the donor-acceptor pair. His prediction that the rate of transfer depends on the inverse sixth power of the distance between the chromophores was verified previously. In this work, we tested a second prediction of F6rster's theory, namely, that the transfer rate is proportional to J, the magnitude of the overlap between the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor.The energy donor was an N-methylindole moiety, and the acceptor was a ketone. These chromophores were fused to a rigid steroid that separated them by 10.2 A. Rate constants for singlet-singlet energy transfer in this system were obtained by nanosecond flash spectroscopy. J was varied over a 40-fold range simply by altering the solvent. We found that the transfer rate is proportional to J, as predicted by F6rster's theory. The results bear on the potential use of this energy transfer process to measure distances in biological macromolecules. It is evident that the length of such a spectroscopic ruler can readily be controlled by varying the magnitude of the spectral overlap integral of the energy donor-acceptor pair.Singlet-singlet energy transfer can occur over distances of the order of 30 A. 13 Forster has proposed that the transfer occurs by a dipole-dipole resonance interaction between the energy donor and acceptor chromophores. In his theory, the rate constant for transfer kT is related to geometric and spectroscopic factors by5 kT = r-JK2 n-4 kF X 8.71 X 1023 sec-1where r is the distance (in A) between the centers of the donor and acceptor transition moments, K2 is the dipole-dipole orientation factor, n is the refractive index of the medium, and kF is the rate constant (in sec1) for fluorescence emission by the donor. The spectral overlap integral J, which measures the extent to which the donor and acceptor transitions are in resonance, is given by = fF(X)e(X)dX (2) where F (X) is the fluorescence intensity of the energy donor at wavelength X (in cm), and E(X) is the molar decadic extinction coefficient (in cm-' M-) of the energy acceptor.

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Energy Transfer. A System with Relatively Fixed Donor-Acceptor Separation

Cited by 195 publications

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Detection of actin assembly by fluorescence energy transfer.

Detection of actin assembly by fluorescence energy transfer.

Enhancement of banding patterns in human metaphase chromosomes by energy transfer

Dependence of the Kinetics of Singlet-Singlet Energy Transfer on Spectral Overlap

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