Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding

Abstract

Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Cooperative DNA Binding by the B-Isoform of Human Progesterone Receptor: Thermodynamic Analysis Reveals Strongly Favorable and Unfavorable Contributions to Assembly

“…This weak interaction translates to only a 5-fold increase in binding affinity, and is reflected in the shallow transition of the PRE 1-binding isotherm. The large error associated with this term arises because intrinsic binding affinities and cooperativity terms are typically highly correlated, making it difficult or impossible to uniquely resolve their values (33). It is only because binding to the PRE is weakly cooperative that we were able to resolve this parameter.…”

The Oct-2 protein binds cooperatively to adjacent octamer sites.

“…The value of kj determined from this ex periment was identical, within experimental error, to that determined from probe 2xOA ( Table 2). As another, but related, test of the validity of the model of coopera tive interactions of protein binding to two identical oc tamer sites, values for k^ and ki2 can be determined by simultaneous analysis of total data from both the singlesite probe experiment and the 2xOA probe experiment Senear et al 1986). The results of this analysis are completely consistent with the analysis of each probe alone (Table 2) and support the conclusion that Oct-2 binding to two adjacent octamer sites show 10-fold cooperativity.…”

Global analysis of non-specific protein–nucleic interactions by sedimentation equilibrium

“…However, nonspecific interactions generally have lower affinity and higher dissociation rates than specific interactions and are thus particularly susceptible to artifacts associated with dissociation during the measurement. In addition, nonspecific interactions typically involve binding of multiple protein ligands, and in the case of the filter-binding assay, there is no defined relationship between the extent of saturation and the degree of filter retention [21]. Therefore, it is advantageous to employ alternative methods that directly probe solution equilibria, such as spectroscopic measurements, calorimetry and analytical ultracentrifugation.…”