Linkage of pH, Anion and Cation Effects in Protein-Nucleic Acid Equilibria: Escherichia coli SSB Protein-Single Stranded Nucleic Acid Interactions

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We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB) 65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering ∼ 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to dimers of tetramers (octamers). The intrinsic association equilibrium constant, K obs, and the "limited" cooperativity parameter, ω T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, ω T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K obs decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K obs on [NaF] is independent of pH with ∂log K obs/∂log[NaF] = -4·5(±0·5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (∂log K obs/∂log[NaCl]) ranges from -12·0(±0·8) at pH 5·5, to -6·0 (±0·5) at pH 9·0 (at 25°C). Thus Cl release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F -. The linkages of ion concentration and pH on K obs can be described in terms of: (1) cation release from the polynucleotide; (2) release of Cl from sites on the SSB tetramer that require protonation to bind Cl -; (3) binding of cations to sites on the SSB tetramer which require deprotonation of cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.