Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.


Abstract

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102. Study holds ProTherm entries: 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 13629, 13630, 13631, 13632, 13633, 13634 Extra Details: cysteine-free pseudo wild type lysozyme, 1L63 (C54T, C97A) T4 lysozyme; alpha-helix; energetic; structural;,hydrophobic core; thermodynamic

Submission Details

ID: kcozXNwS3

Submitter: Connie Wang

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

Version: 1

Publication Details
Dao-pin S;Anderson DE;Baase WA;Dahlquist FW;Matthews BW,Biochemistry (1991) Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. PMID:1747370
Additional Information

Number of data points 60
Proteins Endolysin ; Endolysin ; Endolysin
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: dCp ionic:KCl,EDTA: 25 mM,0.1 mM, temp:47.9 C, pH:10.4 ; Experimental Assay: ddG ionic:KCl,EDTA: 25 mM,0.1 mM, temp:47.9 C, pH:10.4 ; Experimental Assay: dCp temp:61.9 C, ionic:KCl: 25 mM, pH:6.5 ; Experimental Assay: ddG temp:61.9 C, ionic:KCl: 25 mM, pH:6.5 ; Experimental Assay: dCp temp:65.3 C, ionic:KCl: 25 mM, pH:5.3 ; Experimental Assay: ddG temp:65.3 C, ionic:KCl: 25 mM, pH:5.3 ; Experimental Assay: dCp ionic:KCl: 25 mM, temp:64.6 C, pH:4.4 ; Experimental Assay: ddG ionic:KCl: 25 mM, temp:64.6 C, pH:4.4 ; Experimental Assay: dCp pH:3.0, ionic:KCl: 25 mM, temp:51.6 C ; Experimental Assay: ddG pH:3.0, ionic:KCl: 25 mM, temp:51.6 C ; Experimental Assay: dCp pH:10.4, ionic:KCl: 25 mM, details:Additives EDTA (0.1 mM), ; Experimental Assay: Tm pH:10.4, details:Additives EDTA (0.1 mM), ; Experimental Assay: dHvH pH:10.4, details:Additives EDTA (0.1 mM), ; Experimental Assay: dCp ionic:KCl: 25 mM, pH:6.5 ; Experimental Assay: Tm details:Additives , pH:6.5 ; Experimental Assay: dHvH details:Additives , pH:6.5 ; Experimental Assay: dCp ionic:KCl: 25 mM, pH:5.3 ; Experimental Assay: Tm details:Additives , pH:5.3 ; Experimental Assay: dHvH details:Additives , pH:5.3 ; Experimental Assay: dCp ionic:KCl: 25 mM, pH:4.4 ; Experimental Assay: Tm details:Additives , pH:4.4 ; Experimental Assay: dHvH details:Additives , pH:4.4 ; Experimental Assay: dCp pH:3.0, ionic:KCl: 25 mM ; Experimental Assay: Tm pH:3.0, details:Additives ; Experimental Assay: dHvH pH:3.0, details:Additives ; Derived Quantity: dTm pH:10.4, details:Additives EDTA (0.1 mM), ; Derived Quantity: dTm details:Additives , pH:6.5 ; Derived Quantity: dTm details:Additives , pH:5.3 ; Derived Quantity: dTm details:Additives , pH:4.4 ; Derived Quantity: dTm pH:3.0, details:Additives
Libraries Mutations for sequence MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL
Sequence Assay Result Units