Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability.


Abstract

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 13463, 13464, 13465, 13466, 13467, 13468, 13469, 13470, 13471, 13472, 13473, 13474, 13475, 13476, 13477, 13478, 13479, 13480, 13481, 13482, 13483, 13484, 13485, 13486, 13487, 13488 Extra Details: protein structure; electrostatics; lysozyme;,mutagenesis; crystallography

Submission Details

ID: o9ngPtNL4

Submitter: Connie Wang

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

Version: 1

Publication Details
Dao-pin S;Söderlind E;Baase WA;Wozniak JA;Sauer U;Matthews BW,J. Mol. Biol. (1991) Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability. PMID:1942034
Additional Information

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 PDB Entries

Structure ID Release Date Resolution Structure Title
3EML 2008-09-24T00:00:00+0000 2.6 The 2.6 A Crystal Structure of a Human A2A Adenosine Receptor bound to ZM241385.
3SB8 2011-06-03T00:00:00+0000 2.65 Cu-mediated Dimer of T4 Lysozyme D61H/K65H by Synthetic Symmetrization
5JWW 2016-05-12T00:00:00+0000 1.47 T4 Lysozyme L99A/M102Q with 1-Hydro-2-ethyl-1,2-azaborine Bound
3SB9 2011-06-03T00:00:00+0000 2.45 Cu-mediated Dimer of T4 Lysozyme R76H/R80H by Synthetic Symmetrization
5YQR 2017-11-07T00:00:00+0000 2.4 Crystal structure of the PH-like domain of Lam6
1CTW 1999-08-20T00:00:00+0000 2.1 T4 LYSOZYME MUTANT I78A
3SN6 2011-06-28T00:00:00+0000 3.2 Crystal structure of the beta2 adrenergic receptor-Gs protein complex
2RBQ 2007-09-19T00:00:00+0000 1.63 3-methylbenzylazide in complex with T4 L99A/M102Q
1T8F 2004-05-12T00:00:00+0000 2.15 Crystal structure of phage T4 lysozyme mutant R14A/K16A/I17A/K19A/T21A/E22A/C54T/C97A
3CDV 2008-02-27T00:00:00+0000 1.73 Contributions of all 20 amino acids at site 96 to the stability and structure of T4 lysozyme

Relevant UniProtKB Entries

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