Rotational Diffusion of Biological Macromolecules by Time‐resolved Delayed Luminescence (Phosphorescence, Fluorescence) Anisotropy

Jovin, Thomas M.; Bartholdi, Marty F.; Vaz, Winchil L.C.; Austin, Robert H.

doi:10.1111/j.1749-6632.1981.tb20753.x

Cited by 90 publications

(60 citation statements)

References 32 publications

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“…In general, the properties measured for the methylene blue-DNA complex are similar to those of other intercalating § Measured by phase partitioning against 30% pentane/70% pentanol (19) and by fluorescence titration (20 (23). The initial anisotropy measured for free methylene blue in a high viscosity medium (98% glycerol) is 0.25.…”

Section: Resultssupporting

confidence: 60%

Molecular motion of DNA as measured by triplet anisotropy decay.

Hogan¹,

Wang²,

Austin³

et al. 1982

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

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We have used triplet anisotropy decay techniques to measure the internal flexibility and overall rotational motion of DNA, covering a time range from 15 ns to 200 Is.Nearly monodisperse DNA fragments 65-600 base pairs long were studied by using the intercalating dye methylene blue as a triplet probe. We found that the slow end-over-end tumbling of short DNA fragments (s165 base pairs) is as predicted for a rigid rod.As expected, a longer DNA fragment (600 base pairs) experiences slow segmental motion of its helix axis. We found that, at the earliest times, anisotropy decays more rapidly than expected for a rigid rod, suggesting that, when bound, methylene blue monitors fast internal motion of the helix. Since the rod-like end-over-end tumbling of short fragments rules out fast bending motions, we conclude that the fast components of DNA anisotropy decay are due to twisting motion of the helix, occurring with a time constant near 50 ns.Under physiological conditions, B-form DNA is an exceedingly stable structure. Recently, however, evidence has accumulated suggesting that the structure ofthe helix fluctuates substantially (1)(2)(3)(4)(5)(6)(7)(8). It has been shown by NMR techniques (1-4) that the helix experiences large fast motions in the nanosecond time range. Fast motions in the 1-to 100-ns time range have also been seen by fluorescence anisotropy methods (5-7), using as a probe ethidium bromide intercalated in DNA, and by measuring the ESR line shapes of spin-labeled intercalated dyes (8).Here we describe triplet anisotropy decay measurements of DNA internal motions. As in fluorescence anisotropy measurements, we have used an intercalating dye as a probe; however, we have monitored the anisotropy decay of the triplet rather than the singlet state by using methylene blue, a dye with a high triplet yield. Since the triplet lifetime of methylene blue is =100 pus, we can use triplet anisotropy decay techniques to measure motions over a time scale 1,000 times larger than fluorescence methods allow.MATERIALS AND METHODS In time-resolved triplet anisotropy measurements, chromophores bound rigidly to macromolecules are excited by a short pulse of linearly polarized light. For dyes with high triplet yields, a population of oriented triplet-state molecules results, oriented here meaning that the angular distribution of excited dipole moments is not spherically isotropic. Via rotational Brownian motion, the angular distribution will randomize in time, and the time dependence of the return to a spherically isotropic distribution of dipoles can be monitored either by observing the polarization of the triplet-singlet emission (phosphorescence) or by measuring the absorbance anisotropy associated with the triplet state.In the triplet-state anisotropy decay experiments described here, we have monitored the absorbance anisotropy of the depleted singlet state rather than the triplet state directly. The two techniques are interchangeable; however, singlet-state measurements are often more sensitive because of the high...

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Section: Resultssupporting

confidence: 60%

Molecular motion of DNA as measured by triplet anisotropy decay.

Hogan¹,

Wang²,

Austin³

et al. 1982

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

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“…The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X cm2/s. In consideration of the sizes of the membranebound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbriick, M. I n recent years considerable effort has been dedicated to the study of the diffusion of membrane components in model phospholipid bilayer membranes [for reviews see Cherry (1979), Jovin et al (1981), and Vaz et al (1982)]. The purpose of such model membrane studies is primarily to understand the diffusion of membrane components in terms of the physical parameters governing such diffusion in the quasi-two-dimensional lipid bilayer matrix and, eventually, to have a base line to which diffusion studies in the far more complex biological membranes may be compared.…”

supporting

confidence: 57%

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

Vaz¹,

Criado²,

Madeira³

et al. 1982

Biochemistry

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126

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ABSTRACT:The translational diffusion of bovine rhodopsin, the Caz+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein 1 3000/1) in liquidcrystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X cm2/s. In consideration of the sizes of the membranebound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes

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“…Cells in suspension were excited by a laser pulse, and the phosphorescence emission and anisotropy were recorded as a function oftime. This method is well suited to the study ofrotational motions in membranes in the us-ms time range (15,16). The measurements were augmented by lateral diffusion determinations by the method offluorescence photobleaching recovery (FPR) with fluorescent derivatives of EGF and by biochemical determinations of cellular localization and degradation of the hormone with optical and radioisotope techniques.…”

mentioning

confidence: 99%

“…The basic features of the spectrometer used for time-resolved measurements of polarized phosphorescence have been described (15,16). Excitation of labeled samples (cell suspension of 1-10 X 106 cells per ml) was at 515 nm.…”

mentioning

confidence: 99%

Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors.

Zidovetzki¹,

Yarden²,

Schlessinger³

et al. 1981

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

140

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The rotational diffusion ofepidermal growth factor (EGF)-receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the ps time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone-receptor complexes were mobile with rotational correlation times in the range 25-90 As when labeled and measured at 4C. Prolonged incubation and exposure to higher temperatures (23 and 3°C) resulted in longer times up to 350 ass, indicative ofthe progressive formation ofmicroclusters, estimated to contain 10-50 receptors. Upon internalization of the hormone-receptor complexes, visible patches were observed by fluorescence microscopy, and the rotational correlation times were shorter, indicating a decrease in size ofthe dynamic unit. The sign of the rotational relaxation also varied with localization and processing of the hormones. The rate of lateral diffusion of EGF-receptor complexes, measured under similar conditions by fluorescence photobleaching recovery, increased with temperature in contrast to the rotational motion.

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Rotational Diffusion of Biological Macromolecules by Time‐resolved Delayed Luminescence (Phosphorescence, Fluorescence) Anisotropy

Cited by 90 publications

References 32 publications

Molecular motion of DNA as measured by triplet anisotropy decay.

Molecular motion of DNA as measured by triplet anisotropy decay.

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors.

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