Fluorescence In Presence Research Articles

Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides

Fluorescence-based solution methods have been used to study the binding of the trp repressor of Escherichia coli to a series of oligonucleotides bearing all or partial determinants for high affinity specific binding. The tryptophan, salt concentration and competitor DNA dependence of the binding affinities was examined for these targets. Binding to a fluorescein-labeled 20 base-pair hairpin structure oligonucleotide, which contains a palindromic repressor binding site (GAACTAGTTAACTAGTAC) and is known to bind repressor in a 1 : 1 dimer-DNA complex, resulted in a protein concentration-dependent, competable static quenching of fluorescence in presence of co-repressor, l-tryptophan. The affinity recovered from the fits of these intensity profiles at 100 mM KCl was on the order of 4 × 108M−1. In absence of co-repressor an increase in intensity at high repressor concentration (>10−7M) was observed. The salt concentration dependence of the specific binding of the holo-repressor to this oligonucleotide was approximately half as large as what would be predicted by the number of phosphate contacts in the crystal structures of the complex. Repressor binding to the fluorescein-labeled hairpin 20mer was compared with binding to a rhodamine-labeled 36 base-pair oligonucleotide bearing two inverted structural half-sites GNACT separated by an eight base-pair spacer containing none of the natural intervening sequence. The rather low affinity observed for the 36mer revealed that the intervening sequence in the natural operators contains energetic specificity determinants. Binding to a rhodamine-labeled oligonucleotide bearing a completely non-specific sequence was shown to occur over the same concentration range (>100 nM), regardless of tryptophan concentration, whereas binding to sequences bearing partial specificity ratio between 100 and 1000, depending upon the salt concentration. Even in abscence of added KCl, the specificity ratio of trp repressor was greater than 100, implicating a significant free energy contribution from non-electrostatic interaction forces.

Myelin basic protein domains involved in the interaction with actin.

A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

Effects of differently hydrophobic solvents on the aggregation of cationic dyes as measured by quenching of fluorescence and/or metachromasia of the dyes.

Aggregation of some cationic dyes leads to the appearance of their metachromatic spectra and/or quenching of their fluorescence. Ethanol and urea destroy metachromasia and enhance the fluorescence of such dyes by disaggregating them, suggesting hydrophobic bonds to be involved in their aggregation as in the formation of soap micelles or globular proteins. The ability of alcohols to disaggregate cationic dyes has been shown to be increased in the series methanol, ethanol, iospropanol and tertiary-butanol which is the order of increasing hydrophobic character of the alcohols themselves. Dimethyl urea is shown to be more effective than urea in destroying the metachromasia of toluidine blue, thus supporting the idea of hydrophobic bond to be involved in dye aggregation. Rhodamine 6 G undergoes quenching of its fluorescence in presence of a suitable polyanion but it is not metachromatic like acridine orange. Since only some specific cationic dyes undergo spectral changes with aggregation such changes seem to be the secondary effects of aggregation.