Theory of Fluorescence Depolarization by Anisotropic Rotational Diffusion

Chuang, T. J.; Eisenthal, Kenneth B.

doi:10.1063/1.1678194

Cited by 401 publications

(271 citation statements)

References 12 publications

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“…are measures of the extent to which the emission is depolarized by each rotational component, thereby measuring the restriction upon diffusion of the residue reporter (35).…”

Section: Methodsmentioning

confidence: 99%

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Kim¹,

Beard²,

Harvey³

et al. 2003

Journal of Biological Chemistry

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DNA polymerase (pol) ␤ is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between ␣-helix N and the nascent base pair. Kinetic and structural characterization of pol ␤ has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and ␣-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of ␣-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol ␤ were determined. The results revealed that the segmental motion of ␣-helix N was rapid (ϳ1 ns) and far more rapid than the step that limits chemistry. Binding of Mg 2؉ and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in ␣-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within ␣-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of ␣-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol ␤ deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors. DNA polymerase (pol)1 ␤ is a 39-kDa enzyme composed of two distinct domains of 8-and 31-kDa connected by a proteasehypersensitive hinge region. The amino-terminal 8-kDa domain possesses the 5Ј-deoxyribose phosphate lyase activity required to process the 5Ј-terminus in a DNA gap during base excision repair. The polymerase active site is part of the carboxyl-terminal 31-kDa domain (for a review see Ref. 1). The domain and subdomain organization of pol ␤ is illustrated in Fig. 1, A and B. Although pol ␤ appears to have evolved separately from other families of polymerases of known structure (2), it shares many general structural and mechanistic features with other polymerases. The polymerase domain is composed of three functionally distinguishable subdomains. The polymerase catalytic subdomain coordinates two divalent metal cations that assist the nucleotidyl transferase reaction. Two additional subdomains that have primary roles in duplex DNA binding and nascent base pair (nucleoside 5Ј-triphosphate and templating nucleotide) binding border the catalytic subdomain. These subdomains will ...

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“…are measures of the extent to which the emission is depolarized by each rotational component, thereby measuring the restriction upon diffusion of the residue reporter (35).…”

Section: Methodsmentioning

confidence: 99%

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Kim¹,

Beard²,

Harvey³

et al. 2003

Journal of Biological Chemistry

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“…If the diffusion is different for different directions, then the polarization anisotropy will be a sum of up to five exponentials. 5,14 The number of independent decay constants will be reduced if symmetry is present, e.g., if the diffusion constant D is isotropic, as for a spherical molecule, then the polarization anisotropy decays as exp(Ϫ6Dt), so there is only one decay time. For an ellipsoid, there are two diffusion constants, D ʈ for rotation around the symmetry axis ͑i.e., spinning͒ and D Ќ for rotation perpendicular to the symmetry axis ͑tumbling͒.…”

Section: Theorymentioning

confidence: 99%

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] In this picture, the molecules experience a random force due to collisions with the small molecules of the liquid. The correlations in the motion of the solvent molecules are represented by a friction, which is calculated as if the liquid were a structureless continuum.…”

Section: Introductionmentioning

confidence: 99%

Orientational relaxation times of rhodamine 700 in glycerol-water mixtures

Megens¹,

Sprik²,

Wegdam³

et al. 1997

The Journal of Chemical Physics

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We determined orientational relaxation times for rhodamine 700 dye in glycerol-water mixtures using time-resolved fluorescence depolarization. It appears that the orientational relaxation time varies linearly with the viscosity of the solvent between 1 and 60 cP, in accordance with the Perrin-Stokes-Einstein model with stick boundary conditions. Previously others have found that for two anionic dyes in glycerol-water and a cationic dye in glycerol-ethylene glycol mixtures, the orientational relaxation time becomes less sensitive to the viscosity at very high viscosities (Ͼ25 cP at least͒. We discuss the influence of dye and solvent on the relation between orientational relaxation time and viscosity, which suggests that the relaxation time as a function of viscosity can be scaled on a common curve.

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“…However, for none of our samples was a purely single exponential decay observed: in each case the data could be fit to a sum of two exponentials [Eq. (16)] with lifetimes of approximately 2-3 and 20-27 ns. The lifetime of the shorter component (-2-3 ns) was similar to that measured for free ethidium bromide (1.8 ns).…”

Section: A Fluorescence Lifetimesmentioning

confidence: 99%

Torsion and bending of nucleic acids studied by subnanosecond time-resolved fluorescence depolarization of intercalated dyes

Millar

Robbins

Zewail

1982

The Journal of Chemical Physics

159

109

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Subnanosecond time-resolved fluorescence depolarization has been used to monitor the reorientation of ethidium bromide intercalated in native DNA, synthetic polynucleotide complexes, and in supercoiled plasmid DNA. The fluorescence polarization anisotropy was successfully analyzed with an elastic model of DNA dynamics, including both torsion and bending, which yielded an accurate value for the torsional rigidity of the different DNA samples. The dependence of the torsional rigidity on the base sequence, helical structure, and tertiary structure was experimentally observed. The magnitude of the polyelectrolyte contribution to the torsional rigidity of DNA was measured over a wide range of ionic strength, and compared with polyelectrolyte theories for the persistence length. We also observed a rapid initial reorientation of the intercalated ethidium which had a much smaller amplitude in RNA than in DNA.

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Theory of Fluorescence Depolarization by Anisotropic Rotational Diffusion

Cited by 401 publications

References 12 publications

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Rapid Segmental and Subdomain Motions of DNA Polymerase β

Orientational relaxation times of rhodamine 700 in glycerol-water mixtures

Torsion and bending of nucleic acids studied by subnanosecond time-resolved fluorescence depolarization of intercalated dyes

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