Effects of Monovalent Anions on a Temperature-Dependent Heat Capacity Change for Escherichia coli SSB Tetramer Binding to Single-Stranded DNA

Abstract

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (ΔH obs ) and the negative heat capacity change (ΔC p,obs ) for E. coli SSB homotetramer binding to single stranded (ss) DNA. Using isothermal titration calorimetry we have now examined ΔH obs over a much wider temperature range (5°C to 60°C) and as a function of monovalent salt concentration and type for SSB binding to (dT) 70 under solution conditions that favor the fully wrapped (SSB) 65 complex (monovalent salt concentration ≥ 0.20 M). Over this wider temperature range we observe a strongly temperature dependent ΔC p,obs . The ΔH obs decreases as temperature increases from 5°C to 35°C (ΔC p,obs <0), but then increase at higher temperatures up to 60°C (ΔC p,obs >0). Both salt concentration and anion type have large effects on ΔH obs and ΔC p,obs . These observations can be explained by a model in which SSB protein can undergo a temperature and salt dependent conformational transition (below 35°C), the midpoint of which shifts to higher temperature (above 35°C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference, Br -> Cl -> F -, and these anions are then released upon binding ssDNA, affecting both ΔH obs and ΔC p,obs . We conclude that the experimentally measured values of ΔC p,obs for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation, but rather result from a series of temperature dependent equilibria (ion binding, protonation and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.Obtaining an understanding of the molecular origins for stability and specificity of proteinnucleic acid interactions is a complex problem. Such interactions not only involve multiple networks of hydrogen bonds, salt bridges and non-polar interactions, but are also accompanied by the coupled binding and/or release of small molecules, such as protons, salt ions and water. These coupled binding equilibria will generally contribute significantly to the thermodynamics of protein-nucleic acid binding (ΔG°, ΔH° and ΔS°). For this reason, thermodynamic information obtained under only one set of solution conditions is generally of limited utility for understanding the origins of stability and specificity of any macromolecular interaction, including protein-nucleic acid binding. † This research was supported in part by the NIH (R01 GM30498) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe have been studying the thermodynamics of E. coli SSB protein binding to single stranded (ss) nucleic acids (for reviews see (1-3)), a protein that is involved in DNA replication, recombination and repair (4). E.coli SSB protein is a stable homotetramer (4×18,843 Da) (5), that can interact with ssDNA in multiple binding modes, depending on...

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

“…Bio-5-AMP binding to the W223A variant exhibits a nonlinear dependence of binding enthalpy on temperature. Although this phenomenology has been observed in other systems, the explanation for the observation in this instance is beyond the scope of this work (30)(31)(32)(33). Partial proteolysis with subtilisin provides a low-resolution measure of the ligand-induced folding of the ABL with bio-5'-AMP binding to wild type BirA resulting in a 6.5-fold decrease in the pseudo-first order cleavage rate.…”

Energetics of the protein-DNA-water interaction

“…However, the negative heat capacity change associated with the formation of specific protein-DNA complexes could not be completely explained by taking into account only hydration effects [14,17,18]. Other contributions, like the conformational changes of both proteins and nucleic acids accounting for 20% of the total ΔC p [69-72], the modification of the protonation state of the interacting residues [73] and counterion release [74], have been considered. In particular, even if ion release was generally considered to be favorable for complex formation, several studies demonstrated that the negative contribution from ion-molecule electrostatics, rather than the positive entropy given by the ion reorganization, dominates the salt-dependent solvation effects [36,37].…”

Molecular determinants of the interactions between proteins and ssDNA

“…The complexes between SSBs and ssDNA are often very thermodynamically stable (11,12), and their binding relies on the electrostatic interactions between the negatively charged phosphates of the ssDNA and the complementary positively charged residues [lysine (K), arginine (R), or histidine] of the SSBs. The same electrostatic attraction is also required for protein binding to dsDNA (13), with SSBs differentiating ssDNA from its dsDNA competitor on the basis of the intrinsic properties of each, in that dsDNA is significantly less flexible in regards to the spacing and positioning of its negative charges than ssDNA.…”