Abstract
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.
Highlights
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear
We use single-molecule force spectroscopy (SMFS), wherein a single molecule is held under tension by an applied load and its extension is measured as its structure changes in response to the load[28], to investigate the effect of a ligand with anti-prion activity on the folding of the prion protein PrP
The mechanism of anti-prion action has not yet been determined for any of these molecules. Since deciphering how such ligands work could provide clues to the molecular mechanism for conversion of PrPC into PrPSc and help design improved drugs, we investigated the effects of ligand binding on individual PrP molecules using SMFS
Summary
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Single-molecule approaches have been deployed successfully to study protein misfolding and aggregation, for example identifying misfolded states, determining misfolding pathways, detecting transient oligomeric intermediates and exploring the interactions stabilizing amyloid fibrils[7,16,17,18,19,20,21,22] They have started to be applied to unravel the mechanisms of molecular chaperones[23], showing for example that chaperones help correct folding of substrate proteins by unfolding misfolded molecules to give them a new chance to refold, altering the folding rates of domains, and blocking tertiary contacts in the transition state[23,24,25,26,27]. We find that it acts in multiple ways, both stabilizing the native structure and inhibiting the formation of intermolecular interactions that stabilize misfolded aggregates
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