Determination of thermodynamic functions from scanning calorimetry data

Kidokoro, Shun‐ichi; Wada, Akira

doi:10.1002/bip.360260205

Cited by 92 publications

(78 citation statements)

References 7 publications

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“…In addition, the ⌬H cal values of the mutants in the presence of 10 mM CaCl 2 at their T d were found to be 145.1, 134.0, and 131.5 kcal/mol, respectively. The ratios of ⌬H cal /⌬H vH for every mutant was about 0.95, which is also similar to that of the wild type and Q86D/A92D lysozymes (3), indicating two state denaturation (22). The heat capacity changes (⌬C p ) in the denaturation of the mutants were also similar to that of the wild type and Q86D/A92D lysozyme reported previously (3,5).…”

Section: Enzymatic Activities and Casupporting

confidence: 84%

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Structural and Thermodynamic Responses of Mutations at a Ca2+ Binding Site Engineered into Human Lysozyme

Kuroki

1998

Journal of Biological Chemistry

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Structural determinants of Ca2؉ binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition. Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca 2؉ binding lysozymes and ␣-lactalbumins. The effects of these mutations on the stability and Ca 2؉ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures. The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca 2؉ binding affinity, whereas neither point mutant showed Ca 2؉ affinity, indicating that both residues are essential. The further mutation of Ala-83 3 Lys did not affect the Ca

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Section: Enzymatic Activities and Casupporting

confidence: 84%

“…The pH of the sample solution was confirmed before and after each measurement. Calorimetric (⌬H cal ) and van't Hoff enthalpies (⌬H vH ) were calculated by the method of Kidokoro and Wada (22).…”

Section: Methodsmentioning

confidence: 99%

Structural and Thermodynamic Responses of Mutations at a Ca2+ Binding Site Engineered into Human Lysozyme

Kuroki

1998

Journal of Biological Chemistry

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“…The apparent equilibrium constants of denaturation (K) at 850C in Eq. 1 can be obtained from the integration of each DSC curve at various concentrations of Ca2+ (14). By using the value of In Ko = 4.6 (AG = -3.3 kcal/mol, see (20,21).…”

Section: Resultsmentioning

confidence: 99%

Entropic stabilization of a mutant human lysozyme induced by calcium binding.

Kuroki

Kawakita

Nakamura

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The stabilization mechanism of the mutant human lysozyme with a calcium binding site (D86/92) was investigated by using calorimetric approaches. By differential scanning calorimetry, the enthalpy change (Al) in the unfolding of holo-D86/92 was found to be 6.8 kcal/mol smaller than that of the wild-type and apo-D86/92 lysozymes at 85TC.However, the unfolding Gibbs energy change (AG) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85C, indicating a significant decrease of entropy (TAS = 10.1 kcal/mol) in the presence of Ca2 . Subsequently, the Ca2+ binding process in the folded state of the mutant was analyzed by using titration isothermal calorimetry. The binding enthalpy change was estimated to be 4.5 kcal/mol, and AG was -8.1 kcal/mol at 85°C, which indicates that the binding was caused by a large increase in entropy (TAS = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding AG of -4.8 kcal/mol (Al = -2.6 kcal/mol, TAS = 2.2 kcal/mol) at 85°C. Therefore, the major cause of stabilization of holo-D86/92 is the decrease in entropy of the peptide chain due to Ca2+ binding to the unfolded protein.It is known that ligand binding stabilizes a protein (1-3). This kind of stabilization is considered to be very important in biological systems because the stabilization of a protein can be easily regulated by the concentration of ligand in the system (2, 4). The mechanism of the stabilization is stoichiometrically explained by the shift of the folding-unfolding equilibrium to the folded state caused by the higher affinity of ligand to the folded state (5, 6). However, the stabilization mechanism of a protein induced by ligand binding is not so clear from the thermodynamic point of view. The reactions involve not only the change of protein conformation but also the interaction of ligand with water molecules, which makes it complicated to understand the thermodynamic mechanism of the stabilization induced by ligand binding alone. Therefore, the most effective way to clarify the mechanism is the direct analysis ofboth the denaturation and the ligand binding by using calorimetric approaches in conjunction with information of the tertiary structure of a protein. Two kinds of calorimeters are available for this purpose. One is a differential scanning calorimeter used to obtain thermodynamic parameters of protein unfolding (7,8), and the other is a titration calorimeter used to obtain the thermodynamic parameters of binding of a ligand by a protein (9).A mutant human lysozyme (D86/92) that has an engineered Ca2+ binding site (Ka = 5.0 x 106 M-1) was created by replacing both Gln-86 and Ala-92 with aspartic acid (10). This mutant is a suitable model for the investigation of the stabilization mechanism induced by ligand binding because the x-ray structures of the wild-type (11) and the mutant (12) lysozymes have been already solved at 1.8 A. By using both differential scanning calorimetry (DSC) and isothermal differential titration calorimetry (DTC) it wa...

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“…The heat capacity functions were analyzed by double deconvolution (DD) method, which analyzes the heat capacity functions as reported previously [26,27], and the nonlinear least-squares method with the three-state model. The DD method can determine the enthalpy and the Gibbs free energy directly from the heat capacity function of the native (N), denatured (D), and intermediate (I) states of these states in the transition, using the following Eqs.…”

Section: Differential Scanning Calorimetrymentioning

confidence: 99%