![]() Notable examples of the latter include cyclic tetrapyrroles 11, 46 such as phthalocyanines and porphyrins, which have been found to inhibit PrP Sc accumulation in cell culture 46, 47 and protein misfolding cyclic amplification assays 47, as well as to increase the survival times in animal models 11, 48. Examples include sulphonated dyes such as congo red and its derivatives (for example, curcumin), 36, 37 certain polyanions 38, 39, 2-aminothiozoles 40 and various heterocyclic compounds 41, 42, 43, 44, 45. Despite these uncertainties about the central aspects of the molecular basis for prion diseases, however, several putative small-molecule chaperones with anti-prion activity have been discovered using cellular and/or animal models of disease 34, 35. The structure of PrP Sc remains controversial 31, 32, 33, as does the molecular mechanism of the conversion of PrP C (refs 2, 30). The native, cellular form of PrP, rich in α-helices and denoted PrP C, is converted into a toxic, β-rich form, denoted PrP Sc, which has the ability to recruit further PrP C molecules and thereby propagate the disease 29, 30. Misfolding of PrP causes prion diseases such as Creutzfeldt–Jakob disease, scrapie and bovine spongiform encephalopathy. Here 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. However, there has been little single-molecule work to date on pharmacological chaperones, aside from studies of their effects on amyloid stability 22. They have also 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. 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. Single-molecule methods such as fluorescence and force spectroscopy provide a powerful new approach for addressing this question, because their ability to detect rare and transient states, identify and characterize different subpopulations in a heterogeneous ensemble, and follow conformational changes in a single molecule with high resolution 13 is ideally suited to probing misfolding processes 14, 15. Such strategies have yielded a number of compounds with promising potential 11, 12, but it has proven challenging to improve their performance and develop effective therapeutics, in part because the mechanism of action of putative pharmacological chaperones is not known. Such a picture has motivated the development of small-molecule drugs that could act as pharmacological chaperones to promote native folding of disease-related proteins 9, 10. However, this proteostatic machinery is likely overwhelmed in misfolded diseases 5, 6, allowing misfolded protein species-including the prefibrillar oligomers thought to be the most neurotoxic species 1, 7, 8-to accumulate. Misfolding is normally held in check in the cell through the action of molecular chaperones, which help proteins find their native structure, preventing misfolding in the first place 3, or the proteasome, which degrades incorrectly folded products 4. Misfolded proteins are an important feature of many neurodegenerative diseases, from Alzheimer’s and Parkinson’s to amyotrophic lateral sclerosis (ALS) and prionopathies, collecting in characteristic amyloid plaques 1, 2. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. 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. Fe-TMPyP also binds the unfolded state, delaying native refolding. 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. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. 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. The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear.
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