Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.     

DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy

We have used transmission electron microscopy to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding amino acid residues 94-312, to linear double-stranded DNA bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCl at low temperature ( approximately 4 degrees C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induced by p53 varied from approximately 40 degrees to 48 degrees upon decreasing of the KCl concentration from 50 mM to approximately 1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35-37 degrees for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (sigma approximately 39 degrees ) than at high salt concentration (sigma approximately 31 degrees ) or for p53CD (sigma approximately 24-27 degrees ). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.     

Direct observation of the enthalpy change accompanying the native to molten-globule transition of cytochrome c by using isothermal acid-titration calorimetry

The enthalpy change accompanying the reversible acid-induced transition from the native (N) to the molten-globule (MG) state of bovine cytochrome c was directly evaluated by isothermal acid-titration calorimetry (IATC), a new method for evaluating the pH dependence of protein enthalpy. The enthalpy change was 30 kJ/mol at 30 degrees C, pH 3.54, with 500 mM KCl. The results of the global analysis of the temperature dependence of the excess enthalpy from 20 to 35 degrees C demonstrated that the N to MG transition is a two-state transition with a small heat capacity change of 1.1 kJ K(-1) mol(-1). The present findings were also indicative of the pH dependence of the enthalpy and the heat capacity of the MG state, -13 kJ mol(-1) pH(-1) and -1.0 kJ K(-1) mol(-1) pH(-1), respectively, at 30 degrees C within a pH range from 2 to 3.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Magnesium is required for specific DNA binding of the CREB B-ZIP domain

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5′-TGACGTCA-3′), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl₂ and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5′-TAACGTTA-3′), C/EBP (5′-TTGCGCAA-3′) and AP-1 (5′-TGAGTCA-3′) sites. The addition of 10 mM MgCl₂ diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using ‘standard’ EMSA conditions (0.25× TBE), CREB bound all the DNA sequences examined. The CREB–CRE complex had an apparent K_d of ~300 pM, PAR of ~1 nM, C/EBP and AP-1 of ~3 nM and SP1 of ~30 nM. The addition of 10 mM MgCl₂ to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl₂. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.     

The energetics of specific binding of AT-hooks from HMGA1 to target DNA

The interaction of the second and third AT-hooks of HMGA1 (formerly HMGI/Y), which bind selectively in the minor groove of an AT-rich DNA sequence, was studied at different temperatures and ionic strengths by spectropolarimetry, spectrofluorimetry, isothermal titration calorimetry and differential scanning calorimetry. The data show that binding of the ten amino acid core element of the two AT-hooks, which penetrates deep into the minor groove, is entropically driven: both the entropy and enthalpy of association of the peptides to the target DNA are positive up to 50 degrees C. The seven amino acid extension of the core in the second AT-hook, which extends out from the minor groove and loops over the phosphodiester backbone, adds a substantial negative enthalpic component into the binding of the 17 residue DBD2 peptide to DNA that corresponds in magnitude to the enthalpy of formation of two hydrogen bonds. The ionic strength dependence of the association constant allowed an estimation of the electrostatic component of binding and, by subtraction, the contribution of the non-electrostatic component, which results from dehydration of the contacting surfaces and makes up almost 70% of the total energy of complex formation. The exceptionally large positive entropy and enthalpy of association of the core AT-hook peptides with target DNA suggest that the water, which is removed from the minor groove of DNA upon binding, is in a highly ordered state. Acetylation of the lysine residue in the second AT-hook, which corresponds to Lys65 of HMGA1, has little effect on the DNA binding; so it appears that repression of the hIFNbeta gene, which follows this modification, is not a direct result of the abrogation of DNA binding.     

A thermodynamic study on the interaction between magnesium ion and human growth hormone

A thermodynamic study on the interaction between magnesium ion and human growth hormone (hGH) was studied at 27 degrees C in NaCl solution (50 mM) using different techniques. Two techniques of ionmetry using a Mg2+selective membrane electrode and isothermal titration calorimetry were applied to obtain the binding isotherm for hGHMg2+; results obtained by both techniques were found to be in good agreement. There is a set of three identical and noninteracting binding sites for magnesium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 46 microM and -17.7 kJ/mol, respectively. Temperature scanning UV-visible spectroscopy was applied to elucidate the effect of Mg2+ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Magnesium ion binding increased the protein thermal stability by increasing the alpha-helix content as well as decreasing both beta and random coil structures. However, the secondary structural change of the protein returns to its native form, including a small change in the tertiary structure, in high concentrations of magnesium ion.     

DNA-binding cytotoxic alkaloids: comparative study of the energetics of binding of berberine, palmatine, and coralyne

Deoxyribonucleic acid is the site of storage and retrieval of genetic information through interaction with proteins and other small molecules. In the present study, the interaction of two natural cytotoxic protoberberine plant alkaloids, berberine and palmatine, and a synthetic derivative, coralyne, with mammalian herring testis DNA was investigated using a combination of isothermal titration calorimetry, differential scanning calorimetry, and optical melting experiments to characterize the energetics of their binding. The binding constants of these alkaloids to DNA under identical conditions were evaluated from the UV melting data, and the enthalpy of binding was elucidated from isothermal titration studies. The binding constants of berberine, palmatine, and coralyne to DNA were found to be 1.15 x 10(4), 2.84 x 10(4), and 3.5 x 10(6) M(-1) at 20 degrees C in buffer of 20 mM [Na+]. Parsing of the free energy change of the interaction observed into polyelectrolytic and nonpolyelectrolytic components suggested that although these alkaloids are charged, the major contributor of about 75% of the binding free energy arises from the nonpolyelectrolytic forces. The binding in case of palmatine and coralyne was predominantly enthalpy driven with favoring smaller entropy terms, while that of berberine was favored by both negative enthalpy and positive entropy changes. Temperature dependence of the binding enthalpies determined from ITC studies in the range 20-40 degrees C was used to calculate the binding-induced change in heat capacity (DeltaC(o)(p)) values as -117, -135, and -157 cal/mol K, respectively, for berberine, palmatine, and coralyne. Taken together, the results suggest that the DNA binding of the planar synthetic coralyne is stronger and thermodynamically more favored compared to the buckled natural berberine and palmatine.     

Creation of genetic information by DNA polymerase of the archaeon Thermococcus litoralis: influences of temperature and ionic strength.

DNA polymerase of the archaeon Thermococcus litoralis can synthesize a long stretch of linear double-stranded DNA in the complete absence of added primer and template DNAs. This finding suggests that genetic information can potentially be created by protein. We report here the effects of temperature, ionic strength and pH on this ab initio DNA synthesis by the protein in vitro . When the temperature of the reaction was changed, the sequence of the product DNA changed markedly. For instance, the reaction products were (TAAT) n at 69 degrees C, (TATCCGGA) n at 84 degrees C and (TATCGCGATAGCGATCGC) n at 89 degrees C. The ionic strength of the reaction condition also affected the sequence: it was (TATCTAGA) n with 0 mM KCl, (TATATACG) n with 50 mM KCl and (TATAGTTATAAC) n with 100 mM KCl at 74 degrees C. When the pH of the reaction condition was changed from 6.8 to 10.8, the size of the product DNA decreased, but its sequence did not. These results demonstrate that DNA synthesized ab initio by DNA polymerase of T.litoralis is markedly influenced by the reaction conditions. The results also suggest that genetic information that might have been created by protein on the early earth is strongly influenced by environmental factors.     

Calorimetric and spectroscopic investigation of drug-DNA interactions. I. The binding of netropsin to poly d(AT)

We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.     

Cardiac-specific Nkx2.5 homeodomain: conformational stability and specific DNA binding of Nkx2.5(C56S)

The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound to the same specific DNA. The thermal unfolding of Nkx2.5(C56S) at pH 6.0 or 7.4 is a reversible, two-state process with unit cooperativity, as measured by differential scanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) at pH 7.4 increases T(m) from 44 to 54 +/- 0.2 degrees C and DeltaH from 34 to 45 +/- 2 kcal/mol (giving a DeltaC(p) of approximately 1.2 kcal K(-)(1) mol(-)(1) for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at <37 degrees C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2-6 x 10(8) M(-)(1) from 10 to 37 degrees C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of DeltaH gives a DeltaC(p) of -0.18 +/- 0.01 kcal K(-)(1) mol(-)(1). This corresponds to 648 +/- 36 A(2) of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.     

Salt bridge exchange binding mechanism between streptavidin and its DNA aptamer – thermodynamics and spectroscopic evidences

Protein‐nucleic acids binding driven by electrostatic interactions typically are characterized by the release of counter ions, and the salt‐inhibited binding association constant (K_a) and the magnitude of exothermic binding enthalpy (ΔH). Here, we report a non‐classical thermodynamics of streptavidin (SA)–aptamer binding in NaCl (140–350 mM) solutions near room temperatures (23–27 °C). By using isothermal titration calorimetry (ITC) and circular dichroism (CD)/fluorescence spectroscopy, we found that the binding was enthalpy driven with a large entropy cost (ΔH ?20.58 kcal mol^?1, TΔS ?10.99 kcal mol^?1, and K_a 1.08 × 10⁷ M^?1 at 140 mM NaCl 25 °C). With the raise of salt concentrations, the ΔH became more exothermic, yet the K_a was almost unchanged (ΔH ?26.29 kcal mol^?1 and K_a 1.50 × 10⁷ M^?1 at 350 mM NaCl 25 °C). The data suggest that no counter Na⁺ was released in the binding. Spectroscopy data suggest that the binding, with a stoichiometry of 2, was accompanied with substantial conformational changes on SA, and the changes were insensitive to the variation of salt concentrations. To account for the non‐classical results, we propose a salt bridge exchange model. The intramolecular binding‐site salt bridge(s) of the free SA and the charged phosphate group of aptamers re‐organize to form the binding complex by forming a new intermolecular salt bridge(s). The salt bridge exchange binding process requires minimum amount of counter ions releasing but dehydration of the contacting surface of SA and the aptamer. The energy required for dehydration is reduced in the case of binding solution with higher salt concentration and account for the higher binding exothermic mainly. Copyright © 2013 John Wiley & Sons, Ltd.     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.