A General Method of Analysis of Ligand Binding to Competing Macromolecules Using the Spectroscopic Signal Originating from a Reference Macromolecule. Application to Escherichia coli Replicative Helicase DnaB Protein−Nucleic Acid Interactions^,

Abstract

Quantitative and accurate analyses of protein-nucleic acid interactions in solution are greatly facilitated if the formation of the complex is accompanied by a large change of the spectroscopic signal (e.g., fluorescence) originating from the protein or nucleic acid. However, there are many instances when protein-nucleic acid interactions do not induce adequate changes in spectroscopic properties of the interacting macromolecules. We describe the theoretical and experimental aspects of a general method to analyze such protein-nucleic acid interactions. The method is based on quantitative titrations of a reference nucleic acid with the protein in the presence of a competing nucleic acid whose interaction parameters with the protein are to be determined. The Macromolecule Competition Titration (MCT) method allows for the determination of the absolute average binding density and the free protein ligand concentration over a large binding density range, unavailable by other methods, and construction of a model-independent true binding isotherm. Moreover, the determination of the absolute binding density of the ligand on nonfluorescent nucleic acid is independent of a priori knowledge of the binding characteristics of the protein to the reference fluorescent nucleic acid. Although the MCT method is applicable to any type of physicochemical signal that can be used to monitor the binding, we discuss the details of the method as it applies to the analysis monitored by a change in the nucleic acid fluorescence intensity and anisotropy upon binding a ligand. Moreover, the interaction parameters for a given nucleic acid can be determined by using as a reference the long polymer nucleic acid as well as short oligomers. In particular, the analysis is greatly simplified if the short fluorescent nucleic acid fragment, spanning the exact site-size of the complex and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. We have illustrated the MCT method by applying it to the binding of the Escherichia coli DnaB helicase to unmodified, nonfluorescent single-stranded nucleic acids where the interactions are not accompanied by any adequate spectroscopic signal changes. In order to analyze simultaneous binding of a ligand to different competing nucleic acid lattices, we introduced the combined application of the McGhee-von Hippel theory and the Epstein combinatorial approach for the binding of a large ligand to a linear, homogeneous nucleic acid lattice. Our approach allows one to perform a direct fit of the entire experimental isotherm for the protein binding to two competing nucleic acid lattices without resorting to complex numerical calculations.

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

“…Binding of TraI Helicase to ssDNA: Determination of the Site Size and DNA Binding Parameters-The interaction of TraI with a series of unlabeled ssDNA oligomers of different lengths was studied using the MCT method (27). Titration of 50 nM FL-T 17 (fluorescently labeled T 17 ) with TraI in the absence or presence of 200 or 350 nM ssDNA oligomers of lengths ranging from 13 to 63 nucleotides were conducted in standard DNA binding buffer (Fig.…”

“…The binding density and the corresponding concentration of free TraI were calculated from the titration curve in Fig. 2A using the MCT analysis (27). The solid lines are computer fits of the data using Equation 9 under the condition m ϭ 25 or Equation 10.…”

“…Macromolecule Competition Titration (MCT) Method-The binding of TraI to unlabeled DNA oligomers was investigated using the MCT method as described by Jezewska and Bujalowski (27). In brief, a 6FAM-labeled reference DNA oligomer (total concentration, D TR ) was mixed with increasing concentrations of protein in DNA binding buffer in 384-well plates in the presence of a competing unlabeled DNA oligomer (total concentration, D TS ).…”

“…5Ј-TCG GAT CGC-3Ј dsDNA 11 5Ј-TCG GAT CGC AG-3Ј dsDNA 15 5Ј-TCG GAT CGC AGT CAG-3Ј dsDNA 19 5Ј-TCG GAT CGC AGT CAG ACG G-3Ј dsDNA 21 5Ј-TCG GAT CGC AGT CAG ACG GTC-3Ј dsDNA 23 5Ј-TCG GAT CGC AGT CAG ACG GTC AG-3Ј dsDNA 27 5Ј-TCG GAT CGC AGT CAG ACG GTC AGT GAC-3Ј…”

“…3B, was analyzed using the Epstein equation under the condition m ϭ 25 and g ϭ 2. The K int and were determined to be 6.7 (Ϯ0.2) ϫ10 Binding of TraI to dsDNA, Determination of the Site Size and Binding Parameters-We next examined the interaction of TraI with unlabeled dsDNA using the MCT method (27). Titration of 100 nM FL-dsDNA 15 with TraI in the absence or presence of 500 nM unlabeled dsDNA oligomers of length ranging from 9 to 27 bp in buffer A was performed (Fig.…”