Molecular interactions and structure as analysed by fluorescence relaxation spectroscopy

Rigler, Rudolf; Ehrenberg, Måns

doi:10.1017/s003358350000113x

Cited by 125 publications

(47 citation statements)

References 115 publications

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“…A general equation describing the time dependence of anisotropy for non-parallel excitation and emission moments was given (eq. (4.16) of [ 181) for the case of a fluorescent probe undergoing uniaxial rotation on the surface of a much larger sphere that underwent its own slower but independent isotropic rotation. This model, valid for phosphorescence as well as fluorescence, also serves for the uniaxial rotation of a membrane protein (i.e., the probe) if the rotation of the sphere (i.e., the membrane vesicle) is negligible, when the equation becomes:…”

Section: Discussionmentioning

confidence: 99%

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

1979

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Section: Discussionmentioning

confidence: 99%

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

1979

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“…The value Fmin is the fluorescence emission intensity of the oligomer measured at a netropsin concentration beyond which no change in the fluorescence intensity of the oligomer is observed. duplex and netropsin, respectively, and n is the number of binding sites of netropsin for oligonucleotide (Rigler and Ehrenberg, 1973). K is the intrinsic stability constant for each independent binding site following the description of Scatchard (1949).…”

Section: Thermodynamics Of Netropsin Binding To D(ctganpttcag)2mentioning

confidence: 99%

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin

Patel

Berglund

Nilsson

et al. 1992

European Journal of Biochemistry

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Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10' M -' at 25"C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as AG = -29 kJ/mol, A H = -12 kJ/mol and A S = + 55 J . mol-' . K-' at 25°C. These parameters resemble those of the interaction of poly [(dG-dC) . (dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG), is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.Base-sequence-dependent selectivity and specificity in the interaction of DNA with low-molecular-mass ligands, such as anti-tumour drugs, or with macromolecules such as proteins, is an important biological recognition problem. It is important to understand the detailed molecular background of this recognition and to develop new strategies for experimental studies of specific interactions between DNA and binding ligands. This study concerns the interaction between netropsin and a fluorescent DNA oligomer.Netropsin is an established anti-viral anti-tumour antibiotic (Zimmer and Wiihnert, 1986) which, however, is too toxic for clinical use. Its biological activity is considered to be a consequence of its tight binding to double-helical B-DNA.The interaction of netropsin with DNA has involved a variety of experiments. Foot-printing techniques (Taylor et al., 1984; Dervan, 1986) have established that netropsin binds A + T-rich sites of 5 5 1 base pairs in naturally occurring DNA polymers. A preference for the B conformation of the DNA has been established (Zimmer, 1975). Viscosimetric studies indicate that netropsin binding increases the contour length of A + T-rich DNA helices and makes them more rigid (Reinert et al., 1980). The thermal stability of the DNA is enhanced on netropsin binding and combined spectroscopic and calorimetric techniques have shown that the thermodynamics for netropsin binding to A + T-rich sequences are very favourable, involving an entirely enthalpy-driven interaction Correspondence to A. Grislund,

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“…Only a minor change of the fluorescence intensity was observed in this case, but it is apparent from the polarization data that the formation of the complex was Mg2+-dependent. The quantitative evaluation of the polarization data with respect to the binding parameters [33,7] has been attempted with the assumption of two fluorescent species, free and bound tRNA&7. Linear Scatchard plots were (about 2 h).…”

Section: Trna Complexes With Yeast Ribosomes Investigation Of the Bimentioning

confidence: 99%

“…Sensitive physicochemical methods, such as fluorescence, have to be applied in order to obtain more structural and kinetic information. In other systems fluorescence techniques have been widely used because they offer high sensitivity and a wealth of information [6,7]. A pre- Cohn.…”

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Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli

Robertson

Kahan

Wintermeyer

et al. 1977

European Journal of Biochemistry

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The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichiu coli was studied by steady-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA.ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNA!& was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TYC loop.In the past few years a rather detailed picture of the functional aspects of the tRNA-ribosome interaction has been elaborated [l -51. However, the physical basis of the high precision of protein synthesis and the mechanism of the individual steps have not yet been understood. Sensitive physicochemical methods, such as fluorescence, have to be applied in order to obtain more structural and kinetic information. In other systems fluorescence techniques have been widely used because they offer high sensitivity and a wealth of information [6,7]. A pre- Cohn. EtdBr, 2,7-diarnino-lO-ethyl-9-phenyl-phenanthridinium bromideorethidium bromide; t R N A h , a derivative of tRNAPhe in which wyebutine is replaced by ethidium; tRNA~~:Sl6;,,, tRNA"h' in which dihydrourdcil is replaced by ethidium; tRNAP_h;Wye, tRNAPhe lacking wyebutine.

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Molecular interactions and structure as analysed by fluorescence relaxation spectroscopy

Cited by 125 publications

References 115 publications

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin

Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli

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Molecular interactions and structure as analysed by fluorescence relaxation spectroscopy

Cited by 125 publications

References 115 publications

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin

Interactions of Yeast tRNAPhe with Ribosomes from Yeast and Escherichia coli

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Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli