Alanine scanning mutagenesis of the HyHEL-10 paratope
of the HyHEL-10/HEWL complex demonstrates that the energetically
important side chains (hot spots) of both partners are
in contact. A plot of ΔΔGHyHEL-10_mutant
vs. ΔΔGHEWL_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 complex when 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, D32H/K97, shows that
there is a significant energetic penalty when either partner
is replaced with a neutral side-chain amino acid, but the
D32HN/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 the ΔΔG
values of the Tyr → Phe vs. the corresponding Tyr
→ Ala mutations. The nonpolar contacts in the light
chain contribute only about one-half of the total ΔΔG
observed for the Tyr → Ala mutation, while they are
significantly more important in the heavy chain.
Replacement of the N31L/K96 hydrogen bond with
a salt bridge, N31DL/K96, destabilizes the complex
by 1.4 kcal/mol. The free energy of interaction,
ΔΔGint,
obtained from double mutant cycle analysis showed that
ΔΔGint 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 ΔΔG values are analyzed.