The oligopeptide-binding protein OppA provides
a useful model system for studying the physical chemistry
underlying noncovalent interactions since it binds a variety
of readily synthesized ligands. We have studied the binding
of eight closely related tripeptides of the type Lysine-X-Lysine,
where X is an abnormal amino acid, by isothermal titration
calorimetry (ITC) and X-ray crystallography. The tripeptides
fall into three series of ligands, which have been designed
to examine the effects of small changes to the central
side chain. Three ligands have a primary amine as the second
side chain, two have a straight alkane chain, and three
have ring systems. The results have revealed a definite
preference for the binding of hydrophobic residues over
the positively charged side chains, the latter binding
only weakly due to unfavorable enthalpic effects. Within
the series of positively charged groups, a point of lowest
affinity has been identified and this is proposed to arise
from unfavorable electrostatic interactions in the pocket,
including the disruption of a key salt bridge. Marked entropy-enthalpy
compensation is found across the series, and some of the
difficulties in designing tightly binding ligands have
been highlighted.