A Highly Salt-Dependent Enthalpy Change for Escherichia coli SSB Protein−Nucleic Acid Binding Due to Ion−Protein Interactions

Abstract

We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.

Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein

“…Once the contributions to ΔC p from other sources are determined (30,(36)(37)(38), the remaining (excess) heat capacity was used to calculate the contribution to association from the hydrophobic effect (ΔS HE ):…”

“…This suggested that proton or ion linkage were unlikely to contribute to the heat capacity change; nonetheless, potential contributions from these sources were explored by performing titrations of the binding partners in buffers with different enthalpies of ionization (37) and salt concentrations (38,58). The change in enthalpy (ΔH) of tryptophan binding to TRAP at 25 °C was measured in buffers with ionization enthalpies spanning 10 kcal mol -1 (Figure 8).…”

“…(a) There is very little change in the binding enthalpy in titrations of tryptophan into TRAP in the presence of buffers with different enthalpies of ionization (a slope of -0.08 compared to a slope of ∼1 in the case of a single proton uptake (37,71)), demonstrating that changes in protonation state upon binding do not contribute significantly to the observed heat capacity change. (b) Changes in salt concentration show little effect on the association constant (a slope of -0.17), demonstrating that uptake or release of ions also does not contribute significantly to the observed heat capacity change (38,58). Table 1 Temperature dependent thermodynamics for the titration of tryptophan into TRAP a …”

Fluorescence Anisotropy Studies on the Ku-DNA Interaction

“…Then we performed quantitative studies on the effect of salt on the protein-DNA association to obtain information on the number of cations and anions that are released upon formation of the complex as well as an estimate of the electrostatic component of the binding free energy (17)(18)(19). Similar studies had not been previously performed on any protein involved in NHEJ and, more generally, in eukaryotic DNA repair.…”

Crystal structure of a novel single‐stranded DNA binding protein from Mycoplasma pneumoniae

“…SSBs are involved in a number of cellular processes including DNA replication, recombination, repair, transcription, translation, cold shock response and maintenance of telomeres. [6][7][8][9][10][11] They bind and stabilize transiently formed single-stranded DNA (ssDNA) during biological processes, thereby sheltering them from chemical and nuclease breakdown and preventing the formation of unproductive secondary structures. Despite their structural resemblances even with relatively low sequence similarities, there are variations in their quaternary assemblies (tetrameric, trimeric, and dimeric) as well as sizes which range from about 140 to 300 amino acids.…”