Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.

Abstract

To investigate the ability of a protein to accommodate potentially destabilizing amino acid substitutions, and also to investigate the steric requirements for catalysis, proline was substituted at different sites within the long alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4 lysozyme. Of the four substitutions attempted, three yielded folded, functional proteins. The catalytic activities of these three mutant proteins (Q69P, D72P, and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12 degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals isomorphous with wild-type allowing the structure to be determined at high resolution. In the crystal structure of wild-type lysozyme the interdomain alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure the introduction of the proline causes this bend angle to increase to 14 degrees and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal domain relative to the amino-terminal one. Except for the immediate location of the proline substitution there is very little change in the geometry of the interdomain alpha-helix. The results support the view that protein structures are adaptable and can compensate for potentially destabilizing amino acid substitutions. The results also suggest that the precise shape of the active site cleft of T4 lysozyme is not critical for catalysis. Study holds ProTherm entries: 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 13578, 13579, 13580, 13581, 13582, 13583, 13584, 13585 Extra Details: cysteine-free pseudo wild type lysozyme, 1L63 (C54T, C97A) T4 lysozyme; proline; alpha-helix; amino acid substitution;,melting temperature

Submission Details

ID: F2wsMcHk

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Sauer UH;San DP;Matthews BW,J. Biol. Chem. (1992) Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions. PMID:1733941
Additional Information

Study Summary

Number of data points 54
Proteins Endolysin ; Endolysin ; Endolysin
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: activity temp:65.7 C, buffers:Potassium phosphate: 10 mM, pH:5.5 ; Experimental Assay: ddG temp:65.7 C, buffers:Potassium phosphate: 10 mM, pH:5.5 ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:4.0, temp:62.2 C ; Experimental Assay: ddG buffers:Potassium phosphate: 10 mM, pH:4.0, temp:62.2 C ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, temp:57.5 C, pH:3.5 ; Experimental Assay: ddG buffers:Potassium phosphate: 10 mM, temp:57.5 C, pH:3.5 ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:6.5, temp:62.9 C ; Experimental Assay: ddG buffers:Potassium phosphate: 10 mM, pH:6.5, temp:62.9 C ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:6.5, temp:63.4 C ; Experimental Assay: ddG buffers:Potassium phosphate: 10 mM, pH:6.5, temp:63.4 C ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, temp:63.2 C, pH:6.5 ; Experimental Assay: ddG buffers:Potassium phosphate: 10 mM, temp:63.2 C, pH:6.5 ; Experimental Assay: activity buffers:HCl: 10 mM, temp:38.5 C, pH:2.0 ; Experimental Assay: ddG buffers:HCl: 10 mM, pH:2.0, temp:38.5 C ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:5.5 ; Experimental Assay: Tm buffers:Potassium phosphate: 10 mM, pH:5.5 ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:4.0 ; Experimental Assay: Tm buffers:Potassium phosphate: 10 mM, pH:4.0 ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:3.5 ; Experimental Assay: Tm buffers:Potassium phosphate: 10 mM, pH:3.5 ; Experimental Assay: activity buffers:Potassium phosphate: 10 mM, pH:6.5 ; Experimental Assay: Tm buffers:Potassium phosphate: 10 mM, pH:6.5 ; Experimental Assay: activity buffers:HCl: 10 mM, pH:2.0 ; Experimental Assay: Tm buffers:HCl: 10 mM, pH:2.0 ; Derived Quantity: dTm buffers:Potassium phosphate: 10 mM, pH:5.5 ; Derived Quantity: dTm buffers:Potassium phosphate: 10 mM, pH:4.0 ; Derived Quantity: dTm buffers:Potassium phosphate: 10 mM, pH:3.5 ; Derived Quantity: dTm buffers:Potassium phosphate: 10 mM, pH:6.5 ; Derived Quantity: dTm buffers:HCl: 10 mM, pH:2.0
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 UniProtKB Entries

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