Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile.


Abstract

To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. High-resolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the gamma-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein. Study holds ProTherm entries: 1282, 1283, 1284, 1285, 13648, 13649 Extra Details: T4 lysozyme; thermal denaturation; hydrogen bonds;,stability; structural changes

Submission Details

ID: DjR5ZoKF4

Submitter: Connie Wang

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

Version: 1

Publication Details
Grütter MG;Gray TM;Weaver LH;Wilson TA;Matthews BW,J. Mol. Biol. (1987) Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile. PMID:3681997
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