Abstract

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution. Study holds ProTherm entries: 8037, 8038, 8039, 8040, 8041, 8042, 10286 Extra Details: hen egg white lysozyme; monoclonal antibody HyHEL-10;,association dissociation kinetics; multiple alanine mutations

Submission Details

ID: UwLDVwez

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details

Rajpal A;Kirsch JF,Proteins (2000) Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex. PMID:10813830

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