Studying The Binding Properties Of E-coli Single-stranded DNA Binding Protein By Single-molecule Magnetic Tweezers

Single-stranded binding protein(SSB)can bind ssDNA in high affinity.It can also interact with different kinds of proteins,which attend in DNA metabolism.SSB can protect ssDNA from degradation and lead DNA metabolic protein to their working site.E-coli SSB is a representational SSB among all kinds of SSB.It is a homotetramer that each subunit can binds ssDNA in high affinity.E-coli SSB has three stable binding modes,which binds 65 nt ssDNA,56 nt ssDNA and 35 nt ssDNA respectively under different salt concentration.Many researchers tried to study this phenomenon.However,the reaction process and reaction rate of SSB still remain unclear.We use single molecule dynamics combine with single molecule magnetic tweezers pulling experiment to observe and analyse the reaction between SSB and ssDNA.And we find that under a critical force,a combine/dissociation jump happens.We measure this reaction and get the reaction rate and free energy parameters.We calibrate the free energy to exhibit SSB binding process under zero force.We find that SSB binding process consists of rapid combination and slow wrapping,whose binding force are different.We assume that different electronic density among SSB surface leads to this phenomenon.Maybe it is related to the metabolize regulation.We repeat this experiment under different NaCl concentrations.We find that the combine/dissociation jump happens under different NaCl concentrations.SSB reaction rate and critical force change under different NaCl concentrations.Under special NaCl concentration,the reaction rate changes in large scale,which represents different binding mode.We measure the change of reaction free energy.The result shows that the binding process of SSB and ssDNA has a characteristic like first order phase transition,and a inflexion point can be found under some critical NaCl concentration.We can also calculate the binding entropy change and released ion number,and find their change regularity.We conjecture that different characteristics of SSB under different salt solution lead to different SSB binding mode.Our results exhibit the influence of ion concentration to SSB binding process completely.We do a pulling experiment under different salt specis,and find that the SSB binding process and characteristics are different from those under NaCl solution.We consider that this phenomenon can provide proof about the influence of SSB surface electronic density to the binding process of SSB/ssDNA.Our results indicate the difference of SSB binding processes under different salt conditions,and make the details of different SSB binding modes clearer.