Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

Abstract

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx(‡) of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx(‡) can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx(‡), depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers

“…Another unbinding parameter characterized in this work was the distance to the transition state Δ x ‡ , which is correlated with the type of bond and stiffness of the interaction (Stigler & Rief, 2015). We estimated that for both anti‐Sec61‐selected and anti‐Sec72‐selected translocon populations the Δ x ‡ values were significantly different (i.e., ~0.18 and 0.32 nm, respectively).…”

Determination of protein–protein interactions at the single-molecule level using optical tweezers

“…From the previous expression, at zero force, three parameters are obtained: The lifetime of interaction τ 0 , whose reciprocal corresponds to the dissociation kinetic constant, k off . The free energy difference to the transition state Δ G ≠ , which corresponds to the energy barrier for the dissociation process. The distance to the transition state Δ x ≠ , which correlates with the interactions that must be broken to separate the two molecules (Schlierf and Rief, 2005; Tych et al ., 2013; Stigler and Rief, 2015). …”

“…The distance to the transition state Δ x ≠ , which correlates with the interactions that must be broken to separate the two molecules (Schlierf and Rief, 2005; Tych et al ., 2013; Stigler and Rief, 2015).…”

Effects of Ligand Binding on the Energy Landscape of Acyl-CoA-Binding Protein

“…Relatively few proteins have been studied by mechanical manipulation thus far (11,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and even fewer have been studied for the effects of ligand binding (26)(27)(28)(29). A ligand may affect the mechanical properties of a protein through several potential mechanisms involving specific ligandprotein interactions and minor or extensive conformational rearrangements which will modulate the energy landscape.…”