A New Method to Self‐Associate Proteins Using a Cooperative DNA‐binding Protein

Abstract

Many cellular processes depend on proteins that must self‐associate into larger complexes in order to function. This includes membrane receptors that dimerize (form pairs) to transmit transmembrane signals and scaffolding proteins that assemble into structural filaments. This project is developing a new method to experimentally control protein self‐association in order to better study such processes. The method is based on the DNA‐binding protein HAP1 (heme activation protein 1). As an isolated fragment, the DNA binding region of HAP1 is a monomer in solution but binds cooperatively as a dimer to its DNA target site. Using recombinant DNA methods, an 11 kDa fragment of the HAP1 DNA‐binding region was fused to the 43 kDa maltose binding protein (MBP) to test if adding the HAP1 target site DNA could induce dimerization of the fused MBP. The HAP1 DNA‐binding fragment was modified to remove a protease‐sensitive site observed during recombinant expression. In addition, two different lengths of flexible linker were introduced between the MBP and HAP1 proteins to reduce potential steric interference among MBP, HAP1, and bound DNA. Binding of MBP‐HAP1 fusions to DNA was measured by fluorescence anisotropy using 24 base‐pair fluorescein‐labeled DNA duplexes. A titration of the MBP‐HAP1 fusion with 15 residue linker to a labeled target site showed strong binding with half‐maximal binding at 0.3 μM MBP‐HAP1. In addition, the titration was fit with a Hill constant of 2.0 ± 0.3 indicative of cooperative dimer formation on the DNA. Binding to a labeled non‐target DNA was much weaker with half‐maximal binding greater than 2 μM. Gel filtration chromatography of the protease‐sensitive MBP‐HAP1 fusion showed a DNA‐induced shift from monomer to a larger protein‐DNA complex containing at least two proteins per DNA. These tests are being repeated with the protease‐insensitive MBP‐HAP1 fusion. The ability of the HAP1 DNA‐binding region to induce the self‐association of MBP using an inexpensive DNA ligand should be generalizable to other proteins, giving this method wide utility.