Apparent Heat Capacity Change Accompanying a Nonspecific Protein-DNA Interaction. Escherichia coli SSB Tetramer Binding to Oligodeoxyadenylates

Abstract

We have examined the effects of temperature on the equilibrium constant, Kobs, for Escherichia coli SSB tetramer binding to a series of single-stranded (ss) oligodeoxyribonucleotides, dT(pT)n, dC(pC)n, and dA(pA)n (n = 34, 55, and 69) in order to investigate the thermodynamic basis for the strong preference of E. coli SSB (and other SSB proteins) for binding polypyrimidine stretches of ss-DNA. In addition to the expected base-dependent differences in the magnitude of Kobs, we also observe qualitatively different temperature dependencies for the binding of the SSB tetramer to oligodeoxyadenylates. Linear van't Hoff plots are obtained for SSB tetramer binding to dT(pT)n and dC(pC)n, with delta H0obs ranging from -50 to -100 kcal/mol depending on the oligodeoxynucleotide length and salt concentration. In contrast, all van't Hoff plots for SSB tetramer binding to dA(pA)N are distinctly nonlinear with maxima in K(obs) occurring near 25 degrees C, indicative of an apparent large negative change in molar heat capacity (delta C0P,obs < 0). Thus for the SSB-dA(pA)n interaction, delta H0obs and delta S0obs are both highly temperature dependent, but compensate such that delta G0obs is relatively insensitive to temperature. These nonlinear nonlinear van't Hoff plots are not due to coupling of SSB assembly to dA(pA)n binding or to temperature-dependent shifts in the formation of other SSB-DNA binding modes. The nonlinear van't Hoff plots for SSB tetramer binding to dA(pA)n appear to result from the coupling of two processes: (1) the unstacking of the dA(pA)n bases (occurring with delta H0 > 0 and delta C0P = 0) and (2) the binding of SSB to the unstacked DNA (occurring with delta H0 < 0 and delta C0P = 0). Therefore, although each isolated equilibrium occurs with delta C0P approximately 0, the overall equilibrium displays an apparent delta C0P,obs < 0 due to the coupled equilibrium. The binding of SSB to dT(pT)n and dC(pC)n occurs with delta H0 < 0 and delta C0P,obs = 0, since the bases in these ss-DNA molecules do not stack appreciably. These results indicate that a nonspecific protein-DNA interaction can display a large negative apparent delta C0P; however, this effect appears not to be due to the hydrophobic effect, but rather to a temperature-dependent conformational transition in the DNA that is coupled to protein binding. Implications of these observations for other protein-nucleic acid systems are discussed.

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

“…The latter arises from the fact that the pK a value(s) of one or several groups on the ligand (peptide) or macromolecule (Ca 2ϩ -CaM) can change as a result of complex formation, as has been observed in a variety of other systems (45)(46)(47)(48)(49)(50). Hence, the change in the protonation upon binding can be characterized as the difference in the number of protons bound before and after complex formation, ⌬n ϩ .…”

Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: Implications for triplet expansion diseases

“…To isolate the thermodynamic contribution of order in the triplet repeat loop regions of these hairpins, we have compared these hairpins to the similarly constructed hairpin, X[TTT] 6 X. Implicit in the choice of X[TTT] 6 X as a thermodynamic reference is the assumption that the all-T loop resembles, more than any other base combination, an unstructured loop (47,(49)(50)(51)(52)(53)(54). In support of this assumption, we note that the measured enthalpy for X[TTT] 6 X is in good agreement with the enthalpy predicted by nearest-neighbor data (55,56) for the hairpin stem.…”

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

“…We have undertaken systematic studies of E. coli SSB tetramer binding to ssDNA in an attempt to define the molecular bases for the thermodynamic changes associated with this system, especially the large enthalpy and heat capacity changes. We have previously shown significant effects of adenine base stacking (16,27) …”

“…(1)-net burial of nonpolar surfaces upon complexation (hydrophobic effect) (18,(20)(21)(22), (2) -changes in the vibrational modes of the macromolecules and participating water (18,23,26), and (3)-thermodynamic coupling of one or more binding or conformational equilibria to the main binding reaction of interest (16)(17)(18)(19)27,28).…”

“…SSB binding to ssDNA is accompanied by a large and negative ΔH obs (as large as ∼ -150 kcal/mol for binding to (dT) 70 at < 10 mM monovalent salt concentration) as well as a large and negative ΔC p,obs (16,17). The molecular basis for a negative ΔC p,obs in macromolecular interactions has been the subject of much interest and discussion (16-26) and several origins have been proposed:(1)-net burial of nonpolar surfaces upon complexation (hydrophobic effect) (18,(20)(21)(22), (2) -changes in the vibrational modes of the macromolecules and participating water (18,23,26), and (3)-thermodynamic coupling of one or more binding or conformational equilibria to the main binding reaction of interest (16)(17)(18)(19)27,28).Our studies of E. coli SSB binding to ssDNA have shown that temperature dependent coupled equilibria contribute significantly to the observed ΔH obs as well as ΔC p,obs . For example, the ΔC p,obs for SSB binding to oligodeoxyadenylates ((dA) N ) is approximately two-fold larger in magnitude than for binding to oligodeoxycytidylates ((dC) N ) or oligodeoxythymidylates ((dT) N ) (16).…”