Most proteins form specific, "native" three-dimensional structures that are required for them to function properly. This self-assembly process, called folding, is usually very reliable. When folding goes awry, however, non-native structures can result that lead to disease. I will discuss our work on the structural dynamics of the prion protein PrP, which misfolds through an unknown mechanism into an infectious form that causes "mad cow" disease. We use high-resolution optical tweezers to observe the structural dynamics of individual PrP molecules in real time as they either fold natively or misfold and aggregate into larger structures, focusing on the microscopic mechanisms that determine the structural outcome (native or misfolded). Studying isolated PrP molecules, we measured the energy landscape that governs the native folding, showing that PrP folds in a single step without observable intermediates. We also developed new approaches to detect and characterize states that are rarely occupied, thereby discovering that a single PrP molecule can form several types of misfolded structures. Although these misfolded structures were not stable in a single PrP molecule and hence formed only fleetingly, when two PrP molecules were brought together to form a dimer, misfolding instead became dominant-the native structure was no longer observed to form at all.
Deciphering the series of steps leading to the misfolded dimer, we reconstructed the energy landscape for the misfolding and uncovered a key intermediate driving the change in behavior. Finally, we investigated the effect on the folding of a drug with known anti-prion activity. We found that not only does it stabilize the native structure, altering the energy landscape for unfolding, but it also mimics the action of natural "chaperones" in the cell that reduce misfolding by preferentially preventing the formation of stable misfolded structures. Throughout the talk, I will focus on quantitative descriptions of folding as a physical process, showing how single-molecule probes can be used both to test physical theories of folding and to address unsolved problems in biology and medicine.