Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

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.     

Energetics of sequence-specific protein-DNA association: conformational stability of the DNA binding domain of integrase Tn916 and its cognate DNA duplex

Sequence-specific DNA recognition by bacterial integrase Tn916 involves structural rearrangements of both the protein and the DNA duplex. Energetic contributions from changes of conformation, thermal motions and soft vibrational modi of the protein, the DNA, and the complex significantly influence the energetic profile of protein-DNA association. Understanding the energetics of such a complicated system requires not only a detailed calorimetric investigation of the association reaction but also of the components in isolation. Here we report on the conformational stability of the integrase Tn916 DNA binding domain and its cognate 13 base pair target DNA duplex. Using a combination of temperature and denaturant induced unfolding experiments, we find that the 74-residue DNA binding domain is compact and unfolds cooperatively with only small deviation from two-state behavior. Scanning calorimetry reveals an increase of the heat capacity of the native protein attributable to increased thermal fluctuations. From the combined calorimetric and spectroscopic experiments, the parameters of protein unfolding are T(m) = 43.8 +/- 0.3 degrees C, DeltaH(m) = 255 +/- 18 kJ mol(-1), DeltaS(m) = 0.80 +/- 0.06 kJ mol(-1), and DeltaC(p) = 5.0 +/- 0.8 kJ K(-1) mol(-1). The DNA target duplex displays a thermodynamic signature typical of short oligonucleotide duplexes: significant heat absorption due to end fraying and twisting precedes cooperative unfolding and dissociation. The parameters for DNA unfolding and dissociation are DeltaH(m) = 335 +/- 4 kJ mol(-1) and DeltaC(p) = 2.7 +/- 0.9 kJ K(-(1) mol(-1). The results reported here have been instrumental in interpreting the thermodynamic features of the association reaction of the integrase with its 13 base pair target DNA duplex reported in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3481-3491].     

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.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

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.     

Energetics of sequence-specific protein-DNA association: binding of integrase Tn916 to its target DNA

The DNA binding domain of the transposon Tn916 integrase (INT-DBD) binds to its DNA target site by positioning the face of a three-stranded antiparallel beta-sheet within the major groove. Binding of INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry, differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, and fluorescence spectroscopy. The observed heat capacity change accompanying the association reaction (DeltaC(p)) is temperature-dependent, decreasing from -1.4 kJ K(-1) mol(-1) at 4 degrees C to -2.9 kJ K(-1) mol(-1) at 30 degrees C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and the protein-DNA complex are not changing in parallel when the temperature increases and that thermal motions of the protein and the DNA are restricted in the complex. After correction for this effect, DeltaC(p) is -1.8 kJ K(-1) mol(-1) and temperature-independent. However, this value is still higher than DeltaC(p) of -1.2 kJ K(-1) mol(-1) estimated by semiempirical methods from dehydration of surface area buried at the complex interface. We propose that the discrepancy between the measured and the structure-based prediction of binding energetics is caused by incomplete dehydration of polar groups in the complex. In support, we identify cavities at the interface that are large enough to accommodate approximately 10 water molecules. Our results highlight the difficulties of structure-based prediction of DeltaC(p) (and other thermodynamic parameters) and emphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complex but also of the folding thermodynamics of the protein and the DNA alone, which are described in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].     

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.     

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices.

The thermodynamics of self-assembly of a 14 base pair DNA double helix from complementary strands have been investigated by titration (ITC) and differential scanning (DSC) calorimetry, in conjunction with van't Hoff analysis of UV thermal scans of individual strands. These studies demonstrate that thermodynamic characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual "denatured" strands and in the duplex is essential to understand the origins of duplex stability and to derive stability prediction schemes of general applicability. ITC studies of strand association at 293 K and 120 mM Na+ yield an enthalpy change of -73 +/- 2 kcal (mol of duplex)-1. ITC studies between 282 and 312 K at 20, 50, and 120 mM Na+ show that the enthalpy of duplex formation is only weakly salt concentration-dependent but is very strongly temperature-dependent, decreasing approximately linearly with increasing temperature with a heat capacity change (282-312 K) of -1.3 +/- 0.1 kcal K-1 (mol of duplex)-1. From DSC denaturation studies in 120 mM Na+, we obtain an enthalpy of duplex formation of -120 +/- 5 kcal (mol of duplex)-1 and an estimate of the corresponding heat capacity change of -0.8 +/- 0.4 kcal K-1 (mol of duplex)-1 at the Tm of 339 K. van't Hoff analysis of UV thermal scans on the individual strands indicates that single helix formation is noncooperative with a temperature-independent enthalpy change of -5.5 +/- 0.5 kcal at 120 mM Na+. From these observed enthalpy and heat capacity changes, we obtain the corresponding thermodynamic quantities for two fundamental processes: (i) formation of single helices from disordered strands, involving only intrastrand (vertical) interactions between neighboring bases; and (ii) formation of double helices by association (docking) of single helical strands, involving interstrand (horizontal and vertical) interactions. At 293 K and 120 mM Na+, we calculate that the enthalpy change for association of single helical strands is approximately -64 kcal (mol of duplex)-1 as compared to -210 kcal (mol of duplex)-1 calculated for duplex formation from completely unstructured single strands and to the experimental ITC value of -73 kcal (mol of duplex)-1. The intrinsic heat capacity change for association of single helical strands to form the duplex is found to be small and positive [ approximately 0.1 kcal K-1 (mol of duplex)-1], in agreement with the result of a surface area analysis, which also predicts an undetectably small heat capacity change for single helix formation.     

Conformational changes in single-strand DNA as a function of temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 +/- 0.5 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.     

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.     

Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry

The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.     

The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.