An attempt has been made to identify residues in T4 phage lysozyme that may have strained conformations and, by appropriate site-directed replacements, to reduce this strain and thus increase the thermostability of the protein. Valine 131, within alpha-helix 126-134, was identified as a potential candidate. Its side-chain rotational angle, chi 1, differs by approximately 18 degrees from the low-energy trans configuration. In addition, it is largely solvent exposed, yet is held in a rigid conformation. The mutant protein with Val 131 replaced by alanine was constructed and found to have a melting temperature 0.9 degrees C higher than that of wild-type lysozyme at pH 2.8. As a control, the mutant Val 131----Thr was also constructed and its melting temperature was found to be marginally lower than wild type. High-resolution crystal structure determinations of the mutant lysozymes show that their structures are virtually identical with that of wild-type lysozyme, except for the Val----Ala or Val----Thr replacement. Analysis of the different structures suggests that the design of the Val----Ala substitution was, in principle, successful, although the apparent gain in stability caused by reduction in strain is modest and is somewhat offset by the loss of hydrophobic interactions and by entropic effects. The results also help to provide a structural rationalization for the experimental and empirical observations that alanine has a higher helix propensity than valine or threonine. Study holds ProTherm entries: 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 13857, 13858, 13859, 13860, 13861, 13862, 13863, 13864 Extra Details: All measurements are with 0.2M KCl protein stability; protein design; strain
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:17 p.m.
|Number of data points||38|
|Proteins||Endolysin ; Endolysin ; Endolysin|
|Assays/Quantities/Protocols||Experimental Assay: ddG pH:2.83, temp:51.1 C ; Experimental Assay: ddG pH:2.03, temp:41.47 C ; Experimental Assay: Tm pH:2.83 ; Experimental Assay: dHvH pH:2.83 ; Experimental Assay: Tm pH:2.03 ; Experimental Assay: dHvH pH:2.03 ; Derived Quantity: dTm pH:2.83 ; Derived Quantity: dTm pH:2.03|
|Libraries||Mutations for sequence MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL|