Quantifying the binding affinity of a pharmacological chaperone to transient unfolded states of a normally folded protein

Abstract

Binding of ligands to partially or fully unfolded proteins can play a key role in the mechanism of cellular and pharmacological chaperones, facilitating proper folding. However, it is challenging to quantify the binding affinity of ligands for unfolded states in a protein that is normally folded, as the methods standardly used to destabilize the native fold also affect ligand binding. We used single-molecule force spectroscopy to unfold single protein molecules without altering solution conditions and observe interactions of a ligand with unfolded states. Focusing on pentosan polysulfate (PPS), an anti-prion pharmacological chaperone previously shown to interact with both the native and partially or fully unfolded states of the prion protein, we measured the concentration-dependent effects of PPS binding on the conformational dynamics of bank vole prion protein (BvPrP) molecules held in optical tweezers. We found that PPS stabilized certain partially unfolded intermediate states of BvPrP as well as the fully unfolded state. Strikingly, the tendency to bind unfolded states instead of the folded state increased as the PPS concentration was reduced, implying a higher affinity to unfolded states. From the relative amount of binding to unfolded versus folded states, we estimated that PPS bound roughly 100-fold more tightly to unfolded states than to the native state of PrP. These results reinforce the likely importance of unfolded states in prion misfolding and propagation. More generally, they show how binding affinity to transient, unstable states can be estimated.