  Binding of ligands is often crucial for function yet the effects of ligand
binding on the mechanical stability and energy landscape of proteins are
incompletely understood. Here we use a combination of single-molecule optical
tweezers and MD simulations to investigate the effect of ligand binding on the
energy landscape of acyl-coenzyme A (CoA) binding protein (ACBP). ACBP is a
topologically simple and highly conserved four-alpha-helix bundle protein that
acts as an intracellular transporter and buffer for fatty-acyl CoA and is
active in membrane assembly. We have previously described the behavior of ACBP
under tension, revealing a highly extended transition state (TS) located almost
halfway between the unfolded and native states. Here, we performed force-ramp
and force-jump experiments, in combination with advanced statistical analysis,
to show that octanoyl-CoA binding increases the activation free energy for the
unfolding reaction of ACBP without affecting the position of the transition
state along the reaction coordinate. It follows that ligand binding enhances
the mechanical resistance and thermodynamic stability of the protein, without
changing its mechanical compliance. Steered molecular dynamics simulations
allowed us to rationalize the results in terms of key interactions that
octanoyl-CoA establishes with the four alpha-helices of ACBP and showed that
the unfolding pathway is marginally affected by the ligand. The results show
that ligand-induced mechanical stabilization effects can be complex and may
prove useful for the rational design of stabilizing ligands.
