Fluorescence studies of interactions between Escherichia coli valyl-tRNA synthetase and its substrates

Hélène, Claude; Brun, Francine; Yaniv, Moshe

doi:10.1016/0022-2836(71)90251-8

Cited by 119 publications

(18 citation statements)

References 35 publications

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“…This assumption has been independently shown to hold true by circular dichroism measurements (see below). The parameter/f may be obtained either from measurements in the absence of DNA, adapting these values to that obtained in the presence of DNA taking into account the inner filter effect (Héléne et al, 1971), or from measurements in the presence of DNA, but at high ionic strength (we have used 1 M NaCl) assuming a complete dissociation of the complex at this ionic strength. Within experimental error both methods gave the same values, taking into account the slight enhancement (about 3%) of the headpiece fluorescence in 1 M NaCl as compared to the fluorescence in the binding buffer.…”

Section: Resultsmentioning

confidence: 99%

Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Schnarr¹,

Durand²,

Maurizot³

1983

Biochemistry

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The nonspecific interaction of the short headpiece, the NH2-terminal domain of the lac repressor, with natural DNA and alternating polydeoxynucleotides has been studied by means of fluorescence and circular dichroism. The important quenching of the intrinsic tyrosine fluorescence of the headpiece upon complexation has been used for the determination of the binding isotherms under various environmental conditions. By comparison with theoretical binding curves, we have determined a physical site size of three base pairs. The "perturbated" site size as determined from circular dichroism measurements (about four base pairs) is slightly greater. As in the case of the entire lac repressor, the interaction is strongly ionic strength dependent. The plots log Kobsd as a function of log [NaCl] are linear for salt concentrations greater than 50 mM for the interaction with DNA and greater than 25 mM for the interaction with poly[d(G-C)]. From the slopes of the linear parts of these plots, we determine a number of three electrostatic interactions, assuming no anion release from the protein upon complexation. This value is independent of pH. On the contrary, the association constant Kobsd depends on pH. Complexation requires the protonation of one titrating group of the headpiece with a pK value of 6.7 +/- 0.3, probably the residue histidine-29. The finding is in favor of the idea that the lac repressor interacts with DNA via two headpieces as earlier work has shown that the interaction of the lac repressor with DNA requires the protonation of two groups of the protein.

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Section: Resultsmentioning

confidence: 99%

Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Schnarr¹,

Durand²,

Maurizot³

1983

Biochemistry

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“…The equilibrium between stacked and unfolded conformations for the indole-adenine, guanine, or thymine cases indicates AG near zero, comprising negative enthalpy and entropy terms97 as for nucleic acid base stacking in aqueous solution. The results indicate that total fluorescence quenching of the indole of tryptophan in a polypeptide or protein is to be expected (see also ref [24][25][26][27][28] if it comes into close proximity with a base moiety of a nucleic acid or if intercalation occurs. No evidence has been found for a new fluorescent species emitting at longer wavelength than indole in the Ind-Cj-Ade or Ind-Cs-Cyt models at 25 or 3°.…”

Section: Discussionmentioning

confidence: 99%

Synthetic spectroscopic models. XIV. Intramolecular stacking interactions between indole and connected nucleic acid bases. Hypochromism and fluorescence

Mutai¹,

Gruber²,

Leonard³

1975

J. Am. Chem. Soc.

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“…Solution absorbance was always less than 0.11 at the excitation wavelength. Quantum yields ( ) of VU-9 calmodulin were determined by taking L-tryptophan in water as a reference ( = 0.14 at 20 °C) and were corrected to account for the screening effect of scattered light (Héléne et al, 1971). Quantum yields of VU-1 calmodulin were determined similarly by taking l-tyrosine as reference ( = 0.14 at 20 °C).…”

Section: Methodsmentioning

confidence: 99%

Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III

Kilhoffer

Roberts²,

Adibi³

et al. 1989

Biochemistry

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Absorption and fluorescence properties of VU-9 calmodulin, an engineered calmodulin in which a tryptophan residue has been introduced in position 99, have been investigated. Tryptophan 99 fluoresces with a maximum around 348 nm and is easily quenched by fluorescence quenchers such as acrylamide, indicating that the chromophore is in a polar environment and well exposed to the solvent, a location which has been reported previously for tyrosine 99 in mammalian calmodulin [Kilhoffer, M. C., Demaille, J. G., & Gérard, D. (1981) Biochemistry 20, 4407-4414]. The quantum yields of tryptophan 99 were found to be 0.19 in the absence of calcium and 0.15 in its presence. These values indicate that the chromophore is in a particular microenvironment where it is protected from the quenching mechanisms normally occurring in proteins. Steady-state fluorescence polarization measurements indicate that the protein exhibits segmental mobility both in the absence and in the presence of calcium. Binding of calcium decreases the mobility of the chromophore, a good indication for a rigidification of the protein structure. A quite rigid structure of at least the carboxy-terminal part of VU-9 calmodulin in the presence of Ca2+ is also suggested by Förster energy-transfer measurements.

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Fluorescence studies of interactions between Escherichia coli valyl-tRNA synthetase and its substrates

Cited by 119 publications

References 35 publications

Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Synthetic spectroscopic models. XIV. Intramolecular stacking interactions between indole and connected nucleic acid bases. Hypochromism and fluorescence

Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III

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