Negative Cooperativity within Individual Tetramers of Escherichia coil Single Strand Binding Protein Is Responsible for the Transition between the (SSB)₃₅ and (SSB)₅₆DNA Binding Modes

We have examined the binding of the oligonucleotide dT(pT)₃₄to the Escherichia coli SSB protein as a function of NaCl and MgCl₂concentration (25 °C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for dT(pT)₃₄per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration. At 200 mM NaCl, we observe nearly stoichiometric binding of dT(pT)₃₄to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent. However, when the NaCl concentration is lowered, the overall affinity of dT(pT)₃₄for the second site on the SSB tetramer decreases dramatically. At 1.5 mM NaCl, only a single molecule of dT(pT)₃₄can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)₃₄. MgCl₂is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of dT(pT)₃₄. This binding behavior reflects an intrinsic property of the SSB tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between DNA binding sites within the SSB tetramer. This phenomenon is also responsible for the transition between the two SSB—single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer [Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802] Extreme negative cooperativity stabilizes the (SSB)₃₅binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated. At higher salt concentrations, negative cooperativity is reduced with the result that all four SSB subunits can interact with ss DNA, as in the (SSB)₅₆ and (SSB)₆₅binding modes. The possible biological significance of this negative cooperativity is discussed.