Synthesis and characterisation of fluorescent oligonucleotides. Effect of internal labelling on protein recognition

Hagmar, Per; Bailey, Michael F.; Tong, Glenn; Haralambidis, Jim; Sawyer, William H.; Davidson, Barrie E.

doi:10.1016/0304-4165(95)00015-4

Cited by 21 publications

(37 citation statements)

References 32 publications

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“…ESI‐MS has some advantages over filter binding and gel retardation assays for study of protein–DNA interactions in that these techniques necessarily require separation of bound and free mixture components for analysis and this may perturb the equilibrium position (Hagmar et al1995). However, there are also complicating issues in determination of K d values by ESI‐MS.…”

Section: Resultsmentioning

confidence: 99%

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

et al. 2002

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Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 × 10 −9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of Յ2 × 10 −9 M for Tus where A173 was unchanged. This is the first example in which ESI-MS has been used to compare binding affinities of a DNA-binding protein with mutant proteins for specific DNA recognition sequences. It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.

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Section: Resultsmentioning

confidence: 99%

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

et al. 2002

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“…The fluorescein moiety, attached to the DNA via a thiocarbamide function, is drawn in its predominant (dianionic) form at pH 7.4 (41). × 10 5 M -1 cm -1 , which accounts for a 30% reduction in the absorption at this wavelength due to hypochromicity (19).…”

Section: Methodsmentioning

confidence: 99%

Thermodynamics of the Interaction of the Escherichia coli Regulatory Protein TyrR with DNA Studied by Fluorescence Spectroscopy

Bailey¹,

Davidson²,

Haralambidis³

et al. 1998

Biochemistry

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Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR. Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS. The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1. Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature. The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1. Several conclusions were drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1). Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.

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“…Reagent grade sucrose and triethylene glycol were products of Mallinckrodt (Paris, Kentucky) and Aldrich (St. Louis, Missouri), respectively. TyrR was prepared as described previously (Argaet et al, 1994); and its concentration, expressed in terms of dimer, was determined spectrophotometrically at 280 nm on the basis of a molar absorption coefficient of 34,500 M-1 cm-' for monomer (Hagmar et al, 1995).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

“…Also indicated is the site for attachment of fluorescein (F), the probe inserted to allow characterization of the interaction by fluorescence quenching. tuted for thymidine at position 9 from the 5' end (Hagmar et al, 1995). The resultant oligonucleotide was labeled with FITC and purified by HPLC (Hagmar et al, 1995).…”

Section: Oligonucleotide Synthesismentioning

confidence: 99%

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR

Poon¹,

Bailey²,

Winzor³

et al. 1997

Biophysical Journal

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Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.

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Synthesis and characterisation of fluorescent oligonucleotides. Effect of internal labelling on protein recognition

Cited by 21 publications

References 32 publications

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Thermodynamics of the Interaction of the Escherichia coli Regulatory Protein TyrR with DNA Studied by Fluorescence Spectroscopy

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR

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