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Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the hyhel‐10/lysozyme interaction
Author(s) -
Pons Jaume,
Rajpal Arvind,
Kirsch Jack F.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.5.958
Subject(s) - alanine scanning , lysozyme , mutagenesis , mutant , alanine , chemistry , antigen , site directed mutagenesis , muramidase , antibody , microbiology and biotechnology , biophysics , biochemistry , biology , genetics , amino acid , gene
Alanine scanning mutagenesis of the HyHEL‐10 paratope of the HyHEL‐10/HEWL complexdemonstrates that the energetically important side chains (hot spots) of both partners are in contact.A plot of ΔΔ G HyHEL‐10_mutant vs.ΔΔ G hewl_mutant for the five of six interacting side‐chain hydrogen bonds is linear (Slope = 1).Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complexwhen replaced by alanine, but 6 of the 12 HyHEL‐10 paratope amino acids do.Double mutant cycle analysis of the single crystallographically identified salt bridge, D32 H /K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side‐chain amino acid, but the D32 H N/K97M complex is as stable as the WT.The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing theΔΔ G values of the Tyr Phe vs. the corresponding Tyr Ala mutations.The nonpolar contacts in the light chain contribue only about one‐half of the totalΔΔ G observed for the Tyr Ala mutation, while they are significantly more important in the heavy chain.Replacement of the N31 L /K96 hydrogen bond with a salt bridge, N31D L /K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction,ΔΔ G int , obtained from double mutant cycle analysis showed that ΔΔ G int for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL‐10 single mutant.Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large ΔΔ G values are analyzed.