Abstract
The exploration of single-strand DNA-binding protein (SSB)-ssDNA interactions and their crucial roles in essential biological processes lagged behind other types of protein-nucleic acid interactions, such as protein-dsDNA and protein-RNA interactions. The ssDNA binding protein gene product 32 (gp32) of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during the DNA synthesis. To gain deeper insights into the electrostatic conditions influencing the stability of the ssDNA-gp32 molecular complex, like the salt concentration or some metal ions proven to specifically bind to gp32, we employed a method that performs rapid measurements of the DNA-protein stability using an α-Hemolysin (α-HL) protein nanopore. We indirectly probed the stability of a protein-nucleic acid complex by monitoring the dissociation process between the gp32 protein and the ssDNA molecular complex in single-molecular electrophysiology experiments, but also through fluorescence spectroscopy techniques. We have shown that the complex is more stable in 0.5 M KCl solution than in 2 M KCl solution and that the presence of Zn(2+) ions further increases this stability for any salt used in the present study. This method can be applied to other nucleic acid-protein molecular complexes, as well as for an accurate determination of the drug-protein carrier stability.