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

Abstract

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...

Unfolding Kinetics of Bovine Trypsinogen

“…Analysis of the dependence of the reciprocal of the rate constant on [Caz' 1, according to the model developed by Sancho et al (1 991), indicates that native trypsinogen in 4 M GdmCl can bind Caz+ only twofold weaker than in the absence of denaturant. Such binding effects are perhaps not unusual, as unfolded mutated human lysozyme exhibits rather strong affinity for CaL+ (Kuroki et al, 1992). Further, our analysis indicates that liganded trypsinogen unfolds sixfold slower than the Ca-free form (Fig.…”

The denaturation and degradation of stable enzymes at high temperatures

“…These have included the incorporation of metal binding sites [45], elimination of a buried solvent molecule [46] and addition of a peptide β-hairpin [47]. Detailed comparison of mesophilic and extremely thermophilic enzymes has led to suggestions that the elimination of cavities within the enzyme structure and a decrease in the number of surface loops [48] may be ways of achieving greater stability.…”

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

“…We have already found that only the double mutation (Gln-86 3 Asp and Ala-92 3 Asp) in human lysozyme resulted in the formation of a Ca 2ϩ binding site (5). The precise analyses of the stability (3,5,6) and the Ca 2ϩ affinity (7) using calorimetry have also been performed, and the high resolution structural data of the wild type (12) and Ca 2ϩ binding mutant (13) lysozymes from x-ray crystallography are available. Here we show the effect of the subsequent mutations (Gln-86 3 Asp, Ala-92 3 Asp, and Ala-83 3 Lys) on the Ca 2ϩ binding properties, conformational stabilities, and tertiary structures of these mutants.…”

“…How does the introduction of charged residues within a calcium binding site affect the stability and Ca 2ϩ binding behavior? There are some reports describing the effect of mutations within Ca 2ϩ binding sites on the stability of the protein (3,4). Our approach in this paper is to introduce the minimum perturbation by amino acid replacement and determine the effect of these mutations on the stability and the Ca 2ϩ binding function using calorimetry.…”

“…Furthermore, these effects are interpreted in terms of the structural information from x-ray crystallography. For this purpose, we chose human lysozyme because lysozymes are known to be a suitable model for studying protein function and stability (3,(5)(6)(7). More than 90 sequences of chicken type lysozymes and ␣-lactalbumins are in the sequence data base.…”