Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape

Significance Structural transitions in proteins are characterized by the coefficient for intrachain diffusion, D, which determines the transition kinetics and reveals microscopic properties of the interactions governing folding. D has been measured for unfolded proteins and for native folding, but never for misfolding and aggregation, despite the importance of kinetics for driving these processes. We used single-molecule force spectroscopy to observe the misfolding of individual prion protein (PrP) molecules into stable, nonnative dimers. By reconstructing the energy landscape for dimer misfolding, we compared D for misfolding of PrP to that for native folding. Diffusion was 1,000-fold slower for misfolding, reflecting significant additional roughness in the energy landscape and confirming quantitatively the long-held hypothesis that misfolding landscapes are rougher than native landscapes. The timescale for the microscopic dynamics of proteins during conformational transitions is set by the intrachain diffusion coefficient, D. Despite the central role of protein misfolding and aggregation in many diseases, it has proven challenging to measure D for these processes because of their heterogeneity. We used single-molecule force spectroscopy to overcome these challenges and determine D for misfolding of the prion protein PrP. Observing directly the misfolding of individual dimers into minimal aggregates, we reconstructed the energy landscape governing nonnative structure formation. Remarkably, rather than displaying multiple pathways, as typically expected for aggregation, PrP dimers were funneled into a thermodynamically stable misfolded state along a single pathway containing several intermediates, one of which blocked native folding. Using Kramers’ rate theory, D was found to be 1,000-fold slower for misfolding than for native folding, reflecting local roughening of the misfolding landscape, likely due to increased internal friction. The slow diffusion also led to much longer transit times for barrier crossing, allowing transition paths to be observed directly for the first time to our knowledge. These results open a new window onto the microscopic mechanisms governing protein misfolding.

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