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. Study holds ProTherm entries: 1228, 1229, 1230, 1231, 1232, 1233, 13615, 13616, 13617, 13618 Extra Details: additive : EDTA(1 mM), lysozyme; protein structure; protein design; proline;,glycine
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:16 p.m.
|Number of data points||32|
|Proteins||Endolysin ; Endolysin ; Endolysin ; Endolysin|
|Assays/Quantities/Protocols||Experimental Assay: dCp ionic:KCl,EDTA: 100 mM,1 mM, details:Additives , temp:64.7 C, pH:6.5 ; Experimental Assay: ddG temp:64.7 C, pH:6.5 ; Experimental Assay: dCp temp:41.9 C, details:Additives , pH:2.0, ionic:KCl,EDTA: 100 mM,1 mM ; Experimental Assay: ddG temp:41.9 C, pH:2.0 ; Experimental Assay: dCp pH:6.5, ionic:KCl: 100 mM, details:Additives EDTA (1 mM), ; Experimental Assay: Tm pH:6.5 ; Experimental Assay: dHvH pH:6.5 ; Experimental Assay: dCp pH:2.0, ionic:KCl: 100 mM, details:Additives EDTA (1 mM), ; Experimental Assay: Tm pH:2.0 ; Experimental Assay: dHvH pH:2.0 ; Derived Quantity: dTm pH:6.5 ; Derived Quantity: dTm pH:2.0|
|Libraries||Mutations for sequence MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL|