title: Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.
abstract: It is proposed that the stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa----Pro and the other of the form Gly----Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82----Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77----Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute approximately 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.
mesh_terms: D002151:Calorimetry; D004355:Drug Stability; D009113:Muramidase / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
chemical_list: D011506:Proteins; D009113:Muramidase