Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egr‑1 Zinc-Finger Protein with DNA

The transcription factor Egr-1 specifically binds as a monomer to its 9 bp target DNA sequence, GCG­T­G­G­GCG, via three zinc fingers and plays important roles in the brain and cardiovascular systems. Using fluorescence-based competitive binding assays, we systematically analyzed the impacts of all possible single-nucleotide substitutions in the target DNA sequence and determined the change in binding free energy for each. Then, we measured the changes in binding free energy for sequences with multiple substitutions and compared them with the sum of the changes in binding free energy for each constituent single substitution. For the DNA variants with two or three nucleotide substitutions in the target sequence, we found excellent agreement between the measured and predicted changes in binding free energy. Interestingly, however, we found that this thermodynamic additivity broke down with a larger number of substitutions. For DNA sequences with four or more substitutions, the measured changes in binding free energy were significantly larger than predicted. On the basis of these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of such sequences that might functionally sequester Egr-1

Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling

The residence times of molecular complexes in solution are important for understanding biomolecular functions and drug actions. We show that NMR data of intermolecular hydrogen-bond scalar couplings can yield information on the residence times of molecular complexes in solution. The molecular exchange of binding partners via the breakage and reformation of a complex causes self-decoupling of intermolecular hydrogen-bond scalar couplings, and this self-decoupling effect depends on the residence time of the complex. For protein–DNA complexes, we investigated the salt concentration dependence of intermolecular hydrogen-bond scalar couplings between the protein side-chain ¹⁵N and DNA phosphate ³¹P nuclei, from which the residence times were analyzed. The results were consistent with those obtained by ¹⁵N_<i>z</i>-exchange spectroscopy. This self-decoupling-based kinetic analysis is unique in that it does not require any different signatures for the states involved in the exchange, whereas such conditions are crucial for kinetic analyses by typical NMR and other methods