Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive.


Abstract

A number of mutations have been shown previously to stabilize T4 lysozyme. By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (delta delta G = 3.6 kcal/mol). This shows that it is possible to engineer a protein of enhanced thermostability by combining a series of rationally designed point mutations. It is also shown that this stabilization is achieved with only minor, localized changes in the structure of the protein. This is consistent with the observation that the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associated with the constituent single mutants. One of the seven substitutions, Asn116-->Asp, changes a residue that participates in substrate binding; not surprisingly, it causes a significant loss in activity. Ignoring this mutation, there is a gradual reduction in activity as successively more mutations are combined. Study holds ProTherm entries: 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 13740, 13741, 13742, 13743, 13744, 13745, 13746, 13747, 13748, 13749, 13750, 13751, 13752, 13753, 13754, 13755, 13756, 13757, 13758, 13759, 13760, 13761, 13762, 13763, 13764, 13765, 13766, 13767 Extra Details: additivity; genetic engineering; lysozyme; thermostability;,protein stabilization

Submission Details

ID: z4LX2UJW4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:17 p.m.

Version: 1

Publication Details
Zhang XJ;Baase WA;Shoichet BK;Wilson KP;Matthews BW,Protein Eng. (1995) Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive. PMID:8771182
Additional Information

Study Summary

Number of data points 234
Proteins Endolysin ; Endolysin ; Endolysin ; Endolysin ; Endolysin ; Endolysin ; Endolysin ; Endolysin
Unique complexes 15
Assays/Quantities/Protocols Experimental Assay: activity temp:66.51 C, ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid, Sodium acetate: 1.4 mM, 8.6 ; Experimental Assay: dCp temp:66.51 C, ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid, Sodium acetate: 1.4 mM, 8.6 mM ; Experimental Assay: ddG temp:66.51 C, ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid, Sodium acetate: 1.4 mM, 8.6 mM ; Experimental Assay: activity pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4, H3PO4: 17 mM, 3 mM, temp:53.42 C ; Experimental Assay: dCp pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4, H3PO4: 17 mM, 3 mM, temp:53.42 C ; Experimental Assay: ddG pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4, H3PO4: 17 mM, 3 mM, temp:53.42 C ; Experimental Assay: activity ionic:NaCl: 100 mM, temp:30 degree C, pH:5.4, buffers:Acetic acid,Sodium acetate,: 1.4 mM,8 ; Experimental Assay: dCp ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid,Sodium acetate,: 1.4 mM,8.6 mM, ; Experimental Assay: Tm ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid,Sodium acetate,: 1.4 mM,8.6 mM, ; Experimental Assay: dHvH ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid,Sodium acetate,: 1.4 mM,8.6 mM, ; Experimental Assay: activity pH:3.0, ionic:KCl: 25 mM, temp:30 degree C, buffers:KH2PO4,H3PO4,: 17 mM,3 mM, ; Experimental Assay: dCp pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4,H3PO4,: 17 mM,3 mM, ; Experimental Assay: Tm pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4,H3PO4,: 17 mM,3 mM, ; Experimental Assay: dHvH pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4,H3PO4,: 17 mM,3 mM, ; Derived Quantity: dTm ionic:NaCl: 100 mM, pH:5.4, buffers:Acetic acid,Sodium acetate,: 1.4 mM,8.6 mM, ; Derived Quantity: dTm pH:3.0, ionic:KCl: 25 mM, buffers:KH2PO4,H3PO4,: 17 mM,3 mM,
Libraries Mutations for sequence MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
102L 1992-09-29T00:00:00+0000 1.74 HOW AMINO-ACID INSERTIONS ARE ALLOWED IN AN ALPHA-HELIX OF T4 LYSOZYME
103L 1992-09-29T00:00:00+0000 1.9 HOW AMINO-ACID INSERTIONS ARE ALLOWED IN AN ALPHA-HELIX OF T4 LYSOZYME
104L 1992-09-29T00:00:00+0000 2.8 HOW AMINO-ACID INSERTIONS ARE ALLOWED IN AN ALPHA-HELIX OF T4 LYSOZYME
107L 1992-12-17T00:00:00+0000 1.8 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
108L 1992-12-17T00:00:00+0000 1.8 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
109L 1992-12-17T00:00:00+0000 1.85 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
110L 1992-12-17T00:00:00+0000 1.7 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
111L 1992-12-17T00:00:00+0000 1.8 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
112L 1992-12-17T00:00:00+0000 1.8 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME
113L 1992-12-17T00:00:00+0000 1.8 STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO SITES IN T4 LYSOZYME

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Endolysin P00720 ENLYS_BPT4