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: 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.     

Thermodynamics of apocytochrome b5 unfolding.

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).     

Specific DNA binding by the homeodomain Nkx2.5(C56S): detection of impaired DNA or unfolded protein by isothermal titration calorimetry

Titrations of specific 18-bp duplex DNA with the cardiac-specific homeodomain Nkx2.5(C56S) have utilized an ultrasensitive isothermal titration calorimeter (ITC). As the free DNA nears depletion, we observe large apparent decreases in the binding enthalpy when the DNA is impaired or when the temperature is sufficiently high to produce some unfolding of the free protein. Either effect can be attributed to refolding of the biopolymer that occurs as a result of stabilization due to the large favorable change in free energy on the homeodomain binding to DNA (-49.4 kJ/mol at 298 K). In either case, thermodynamic parameters obtained in such ITC experiments are unreliable. By using a lower temperature (85 vs. 95 degrees C) during the annealing of complementary DNA strands, damage of the 18-bp duplex DNA (T(m) = 72 degrees C) is avoided, and titrations with the homeodomain are normal at temperatures from 10 to 40 degrees C when >95% of the protein is folded. Under the latter conditions, the heat capacity plot is linear with a DeltaC(p) value of -0.80 +/- 0.03 kJ K(-1) mol(-1), which is more negative than that calculated from the burial of solvent accessible surface areas (-0.64 +/- 0.05 kJ K(-1) mol(-1)), consistent with water structures being at the protein-DNA interfaces.     

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.     

Stability of recombinant Lys25-ribonuclease T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Thermodynamics of the binding of Streptomyces subtilisin inhibitor to alpha-chymotrypsin

The binding of Streptomyces subtilisin inhibitor (SSI) to alpha-chymotrypsin (CT) (EC 3.4.21.1) was studied by isothermal and differential scanning calorimetry at pH 7.0. Thermodynamic quantities for the binding of SSI to the enzyme were derived as functions of temperature from binding constants (S. Matsumori, B. Tonomura, and K. Hiromi, private communication) and isothermal calorimetric experiments at 5-30 degrees C. At 25 degrees C, the values are delta G degrees b = -29.9 kJ mol-1, delta Hb = +18.7 (+/- 1.3) kJ mol-1, delta S degrees b = +0.16 kJ K-1 mol-1, and delta C p,b = -1.08 (+/- 0.11) kJ mol-1. The binding of SSI to CT is weak compared with its binding to subtilisin [Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202; Takahashi, K., & Fukada, H. (1985) Biochemistry 24, 297-300]. This difference is due primarily to a less favorable enthalpy change in the formation of the complex with CT. The hydrophobic effect is presumably the major source of the entropy and heat capacity changes which accompany the binding process. The unfolding temperature of the complex is about 7 degrees C higher than that of the free enzyme. The enthalpy and the heat capacity changes for the unfolding of CT were found to be 814 kJ mol-1 and 17.3 kJ K-1 mol-1 at 49 degrees C. The same quantities for the unfolding of the SSI-CT complex are 1183 kJ mol-1 and 39.2 kJ K-1 mol-1 at 57 degrees C.     

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.     

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.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

Inhibition of β-Amylase Activity by Calcium,Magnesium and Zinc Ions Determined by Spectrophotometry and Isothermal Titration Calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH=4.8 (sodium acetate 16?mM) and T=300?K. The Ki and enthalpy of binding for calcium (13.4, 13.1?mM and -14.3?kJ/mol), magnesium (18.6, 17.8?mM and -17.7?kJ/mol) and zinc (17.5, 17.7?mM and -20.0?kJ/mol) were found by spectrophotometric and ITC methods respectively.     

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.     

Reversible unfolding of beta-sheets in membranes: a calorimetric study

The hexapeptide acetyl-Trp-Leu(5) (AcWL(5)) has the remarkable ability to assemble reversibly and spontaneously into beta-sheets on lipid membranes as a result of monomer partitioning followed by cooperative assembly. This system provides a unique opportunity to study the thermodynamics of protein folding in membranes, which we have done using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The results, which may represent the first example of reversible thermal unfolding of peptides in membranes, help to define the contribution of hydrogen bonding to the extreme thermal stability of membrane proteins. ITC revealed that the enthalpy change for partitioning of monomeric, unstructured AcWL(5) from water into membranes was zero within experimental error over the temperature range of 5 degrees C to 75 degrees C. DSC showed that the beta-sheet aggregates underwent a reversible, endothermic, and very asymmetric thermal transition with a concentration-dependent transition temperature (T(m)) in the range of 60 degrees C to 80 degrees C. A numerical model of nucleation and growth-dependent assembly of oligomeric beta-sheets, proposed earlier to describe beta-sheet formation in membranes, recreated remarkably well the unusual shape and concentration-dependence of the transition peaks. The enthalpy for thermal unfolding of AcWL(5) beta-sheets in the membrane was found to be about 8(+/-1)kcal mol(-1), or about 1.3(+/-0.2)kcal mol(-1) per residue.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).