Binding process between E.coli SSB and ssDNA by single-molecule dynamics

Abstract

Single-stranded DNA binding proteins (SSBs) widely exist in different kinds of creatures. It can bind single-stranded DNA (ssDNA) with high affinity. The binding is sequence independent. SSB can also interact with different kinds of proteins, and thus leading them to work at the special sites. It plays an essential role in cell metabolism. E.coli SSB is a representative of SSB among all kinds of SSBs, it is a homotetramer consisting of four 18.9 kD subunits, the homotetramer is stable under low concentration. E.coli SSB has different binding modes under different salt concentrations (for example NaCl). When NaCl concentration is higher than 200 mM, E.coli SSB can bind 65 nt ssDNA, when NaCl concentration is lower than 20 mM, it can bind 35 nt ssDNA, and when the NaCl concentration is between 20 mM and 200 mM, it can bind 56 nt ssDNA. The characteristics of E.coli SSB are so attractive that a large number of researches have been done to distinguish its binding process. Earlier researchers tried to use stop flow technology to study the interaction between SSB and ssDNA in bulk. However, the high affinity between SSB and ssDNA makes this interaction too rapid to be observed at all, and the dissociate interaction even could not be measured. Single molecule technology which combines with low and accurate force offers researchers another way to achieve this goal. Some researchers observed the unwrapping phenomenon in an optical tweezers pulling experiment. However, they did not find the detailed process of binding or dissociation. In our work, we use a magnetic tweezer to pull the SSB/ssDNA complex and find a special phenomenon like double-state jump. Using the single molecule dynamics to analyse the data, we find that this phenomenon is the combination and dissociation between SSB and ssDNA. After comparing the pulling curve of ssDNA only and SSB/ssDNA complex, we find that the SSB binding process consists of two stages, one is rapid combination/dissociation under the action of a critical force; the other is continuous wrapping following the reduced force. According to Bell formula and SSB/ssDNA complex binding model, we obtain the interaction rate and free energy parameters under 0 pN, and we calibrate the free energy to obtain its continuous wrapping part, so we can obtain the whole free energy landscape and understand the binding process. Our analysis way is also applicable to the case of similar interactions to obtain their interaction details and free energy characteristics.