Equilibrium Binding of Escherichia coli Single-Strand Binding Protein to Single-Stranded Nucleic Acids in the (SSB)₆₅ Binding Mode. Cation and Anion Effects and Polynucleotide Specificity

The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(d A), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)₆₅binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model (“tetramer/octamer” model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, K_obsd, and cooperativity parameter, ω_T/_O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)₆₅binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of ω_{T/ O}= 410 ± 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25 °C, pH 8.1). However, K_obsddecreases steeply with increasing salt concentration, such that ∂ log K_obsd/∂ log [NaCl] = -7.4 ± 0.5 for poly(U), -6.1 ± 0.6 for poly(dA), and -6.2 ± 0.3 for poly(A) (25.0 °C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure ∂ log K_obsd/ϑ log [NaBr] = -5.7 ± 0.7, with a lower value of ω_T/O= 130 ± 70. The polynucleotide specificity of the (SSB)₆₅binding mode (0.20 M NaCl, 25.0 °C, pH 8.1) is K_Obsd(dT) > K_obsd(dC) » K_obsd(ss M13 DNA) > K_obsd(I) > K_obsd(U) = 8K_obsd(dA) = 87K_obsd(A) » K_obsd(C). A dramatic effect of anion type on both the salt dependence and magnitude of K_obsdis also observed such that for poly(U) ∂ log K_obsd/∂ log [potassium glutamate] = -5.7 ± 0.4, ∂ log K_obsd/∂ log [NaF] = -4.3 ± 0.4, ∂ log K_obsd/∂ log [NaCH₃COO] = -6.5 ± 0.2, and ∂ log K_obsd/∂ log [NaBr] = -6.7 ± 0.6; in 0.35 M monovalent cation, K_obsd(Glu) = 5.6K_obsd(F) = 11K_obsd(CH₃COO) = (1.1 X 10³)K_obsd(Cl) = (1.1 X 10⁴)K_obsd(Br). These data indicate that significant electrostatic interactions occur in the (SSB)₆₅complexes formed with all polynucleotides, resulting in a net release of both cations and anions, although there are also contributions due to cation and anion uptake.