Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a highresolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into offpathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.protein folding | optical tweezers

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.kinetics | optical trapping | single molecule F olding free-energy landscapes contain, in principle, all the information needed to describe the conformational dynamics of a protein, from the folding kinetics to the locations of energy barriers and the existence of intermediates or nonnative pathways (1). The competition between potential energy and entropy within the funnel-like landscape of most proteins typically allows the full landscape to be reduced to a one-dimensional free-energy profile along the reaction coordinate (2, 3). Folding rates may then be described using Kramers theory (4) in terms of diffusion across a barrier of height ΔG ‡ , where the barrier represents the bottleneck formed by the transition state ensemble. Kramers theory provides a physical derivation of the rates in terms of the shape of the energy profile ( Fig. 1):D is the diffusion constant over the barrier, κ w is the stiffness (curvature) of the potential well, κ b is the stiffness of the barrier, and k B is the Boltzmann constant. The time required to cross over the barrier, the transition path time τ tp , may also be found from the landscape profile (5-7): For an harmonic barrier with ΔG ‡ > 2 k B T (6),

Free-energy-landscape formalisms provide the fundamental conceptual framework for physical descriptions of how proteins and nucleic acids fold into specific three-dimensional structures 1,2 . Although folding landscapes are difficult to measure experimentally, recent theoretical work by Hummer and Szabo 3 has shown that landscape profiles can be reconstructed from non-equilibrium single-molecule force spectroscopy measurements using an extension of the Jarzynski equality 4 . This method has been applied to simulations 5,6 and experiments 7,8 but never validated experimentally. We tested it using force-extension measurements on DNA hairpins with distinct, sequence-dependent folding landscapes. Quantitative agreement was found between the landscape profiles obtained from the non-equilibrium reconstruction and those from equilibrium probability distributions 9 . We also tested the method on a riboswitch aptamer with three partially folded intermediate states, successfully reconstructing the landscape but finding some states difficult to resolve owing to low occupancy or overlap of the potential wells. These measurements validate the landscape-reconstruction method and provide a new test of non-equilibrium work relations.Folding-landscape formalisms have broad applications in biophysics, from improving predictive structural models and protein engineering 10,11 to providing crucial insights into biomolecular structure, dynamics, function and disease 12,13 . Specific characteristics of folding landscapes, such as the roughness of the free-energy surface 14 or the properties of partially folded intermediate 15 and transition states 16 , including how they are altered by temperature changes, solvent substitutions or mutations 17 , have been widely studied by experiment and theory. However, it has proven remarkably challenging to go beyond such isolated features and measure the entire profile of the free-energy landscape along the reaction coordinate.Single-molecule force spectroscopy provides a unique window into folding reactions because of its ability to measure properties of the free-energy landscape. In single-molecule force spectroscopy, a single molecule is held under mechanical tension by a spring-like force probe such as an optical trap or atomic force microscope, and the end-to-end extension of the molecule is recorded while the molecule folds/unfolds under the influence of the denaturing force (Fig. 1a). By this means, the folding energies and rates, partially folded intermediates and similar characteristics may be explored 18 . Measurements can be made either in the equilibrium regime (for example, by maintaining a constant force using an active 19 1 Department of Physics, University of Alberta, 11322-89 Ave, Edmonton AB, T6G 2G7, Canada, 2 National Institute for Nanotechnology, 11421 Saskatchewan Dr, Edmonton AB, T6G 2M9, Canada. † These authors contributed equally to this work. *e-mail: Michael.woodside@nrc-cnrc.gc.ca. or passive 20 force clamp) or in the non-equilibrium regime (for example, by ramping the ...

The formation of selective surface patch binding induced complex coacervates between polyions, chitosan (cationic polyelectrolyte), and alkali-processed gelatin (polyampholyte), both carrying similar net charge, was investigated for two volumetric mixing ratios: r = [chitosan]/[gelatin] = 1:5 and 1:10. Formation of soluble intermolecular complexes between gelatin and chitosan molecules was observed in a narrow range of pH, though these biopolymers had the same kind of net charge, which was evidenced from electrophoretic measurement. This clearly established the role played by selective surface patch binding driven interactions. The temperature sweep measurements conducted on these coacervate samples through rheology and differential scanning calorimetry (DSC) studies yielded two characteristic melting temperatures located at approximately 68 +/- 3 degrees C and 82 +/- 3 degrees C. In the flow mode, the shear viscosity (eta) of the coacervate samples was found to scale with (power-law model) applied shear rate (gamma*) as eta(gamma*) approximately (gamma*)(-k); this yielded k = 0.76 +/- 0.2 (1 s(-1) < gamma* < 100 s(-1)), indicating non-Newtonian behavior. The static structure factor (I(q)) deduced from small angle neutron scattering (SANS) data in the low q (q is the scattering wavevector) (0.018 A(-1) < q < 0.072 A(-1)) region was fitted to the Debye-Bueche regime, I(q) approximately 1/(1 + zeta(2)q(2))2 that yielded a size of zeta approximately 215 +/- 20 A (for r = 1:10) and zeta approximately 260 +/- 20 A (for r = 1:5) samples, implying change in the size of inhomogeneities present with mixing ratio. In the intermediate q region, called the Ornstein-Zernike regime, I(q) approximately 1/(1 + xi(2)q(2)) gave a correlation length of xi approximately 10.0 +/- 2.0 A independent of the mixing ratio. The results taken together imply the existence of a weakly interconnected and heterogeneous network structure inside the coacervate phase separated by domains of polymer-poor regions.

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.