Thermodynamic effects of mutations on the denaturation of T4 lysozyme.


Abstract

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family. Study holds ProTherm entries: 7424, 7425, 7426, 7427, 7428, 7429, 7430, 7431, 7432, 7433, 7434, 7435, 7436, 7437, 7438, 7439, 7440, 7441, 7442, 7443, 7444, 7445, 7446 Extra Details: additive : EDTA(1 mM),cysteine-free pseudo wild type lysozyme, 1L63 (C54T, C97A) alpha-helix; the alanine insertion mutations;,pseudo-wild-type; cooperativity; two-subdomain structure

Submission Details

ID: uX6CaZYP3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Carra JH;Murphy EC;Privalov PL,Biophys. J. (1996) Thermodynamic effects of mutations on the denaturation of T4 lysozyme. PMID:8889173
Additional Information

Number of data points 55
Proteins Endolysin ; Endolysin
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: dHcal pH:4.0, prot_conc:0.659 mg/ml ; Experimental Assay: Tm pH:4.0, prot_conc:0.659 mg/ml ; Experimental Assay: dHcal prot_conc:0.624 mg/ml, pH:3.5 ; Experimental Assay: Tm prot_conc:0.624 mg/ml, pH:3.5 ; Experimental Assay: dHcal pH:3.0, prot_conc:1.10 mg/ml ; Experimental Assay: Tm pH:3.0, prot_conc:1.10 mg/ml ; Experimental Assay: dHcal pH:4.0, prot_conc:1.17 mg/ml ; Experimental Assay: Tm pH:4.0, prot_conc:1.17 mg/ml ; Experimental Assay: dHcal prot_conc:1.16 mg/ml, pH:3.7 ; Experimental Assay: Tm prot_conc:1.16 mg/ml, pH:3.7 ; Experimental Assay: dHcal pH:3.6, prot_conc:1.16 mg/ml ; Experimental Assay: Tm pH:3.6, prot_conc:1.16 mg/ml ; Experimental Assay: dHcal pH:3.5, prot_conc:1.16 mg/ml ; Experimental Assay: Tm pH:3.5, prot_conc:1.16 mg/ml ; Experimental Assay: dHcal pH:3.3, prot_conc:1.16 mg/ml ; Experimental Assay: Tm pH:3.3, prot_conc:1.16 mg/ml ; Experimental Assay: dHcal prot_conc:1.15 mg/ml, pH:3.2 ; Experimental Assay: Tm prot_conc:1.15 mg/ml, pH:3.2 ; Experimental Assay: dHcal prot_conc:1.03 mg/ml, pH:3.0 ; Experimental Assay: Tm prot_conc:1.03 mg/ml, pH:3.0 ; Experimental Assay: dHcal prot_conc:1.11 mg/ml, pH:4.0 ; Experimental Assay: Tm prot_conc:1.11 mg/ml, pH:4.0 ; Experimental Assay: dHcal prot_conc:0.565 mg/ml, pH:3.7 ; Experimental Assay: Tm prot_conc:0.565 mg/ml, pH:3.7 ; Experimental Assay: dHcal pH:3.6, prot_conc:0.997 mg/ml ; Experimental Assay: Tm pH:3.6, prot_conc:0.997 mg/ml ; Experimental Assay: dHcal pH:3.3, prot_conc:1.10 mg/ml ; Experimental Assay: Tm pH:3.3, prot_conc:1.10 mg/ml ; Experimental Assay: dHcal pH:3.2, prot_conc:1.00 mg/ml ; Experimental Assay: Tm pH:3.2, prot_conc:1.00 mg/ml ; Experimental Assay: dHcal prot_conc:0.99 mg/ml, pH:3.0 ; Experimental Assay: Tm prot_conc:0.99 mg/ml, pH:3.0 ; Experimental Assay: dHcal pH:3.7, prot_conc:1.18 mg/ml ; Experimental Assay: Tm pH:3.7, prot_conc:1.18 mg/ml ; Experimental Assay: dHcal prot_conc:0.86 mg/ml, pH:3.5 ; Experimental Assay: Tm prot_conc:0.86 mg/ml, pH:3.5 ; Experimental Assay: dHcal pH:3.3, prot_conc:0.93 mg/ml ; Experimental Assay: Tm pH:3.3, prot_conc:0.93 mg/ml ; Experimental Assay: dHcal pH:3.0, prot_conc:0.89 mg/ml ; Experimental Assay: Tm pH:3.0, prot_conc:0.89 mg/ml ; Experimental Assay: dHcal pH:2.8, prot_conc:1.00 mg/ml ; Experimental Assay: Tm pH:2.8, prot_conc:1.00 mg/ml ; Experimental Assay: dHcal pH:2.6, prot_conc:0.98 mg/ml ; Experimental Assay: Tm pH:2.6, prot_conc:0.98 mg/ml ; Experimental Assay: dHcal pH:2.4, prot_conc:1.01 mg/ml ; Experimental Assay: Tm pH:2.4, prot_conc:1.01 mg/ml ; Derived Quantity: dTm prot_conc:0.624 mg/ml, pH:3.5 ; Derived Quantity: dTm pH:3.0, prot_conc:1.10 mg/ml ; Derived Quantity: dTm prot_conc:1.16 mg/ml, pH:3.7 ; Derived Quantity: dTm pH:3.5, prot_conc:1.16 mg/ml ; Derived Quantity: dTm pH:3.3, prot_conc:1.16 mg/ml ; Derived Quantity: dTm prot_conc:1.03 mg/ml, pH:3.0 ; Derived Quantity: dTm prot_conc:0.565 mg/ml, pH:3.7 ; Derived Quantity: dTm pH:3.3, prot_conc:1.10 mg/ml ; Derived Quantity: dTm prot_conc:0.99 mg/ml, pH:3.0
Libraries Mutations for sequence MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL
Sequence Assay Result Units