No abstract

We propose approximations for force-extension dependencies for the freely jointed chain (FJC) and worm-like chain (WLC) models as well as for extension-force dependence for the WLC model. Proposed expressions show less than 1% relative error in the useful range of the corresponding variables. These results can be applied for fitting force-extension curves obtained in molecular force spectroscopy experiments. Particularly, they can be useful for cases where one has geometries of springs in series and/or in parallel where particular combination of expressions should be used for fitting the data. All approximations have been obtained following the same procedure of determining the asymptotes and then reducing the relative error of that expression by adding an appropriate term obtained from fitting its absolute error. * rafayel.petrosyan@alumni.ethz.ch 2

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

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Folding of proteins and nucleic acids involves a diffusive search over a multidimensional conformational energy landscape for the minimal-energy structure. When examining the projection of conformational motions onto a one-dimensional reaction coordinate, as done in most experiments, the diffusion coefficient D is generally position dependent. However, it has proven challenging to measure such position-dependence experimentally. We investigated the position-dependence of D in the folding of DNA hairpins as a simple model system in two ways: first, by analyzing the round-trip time to return to a given extension in constant-force extension trajectories measured by force spectroscopy, and second, by analyzing the fall time required to reach a given extension in force jump measurements. These methods yielded conflicting results: the fall time implied a fairly constant D, but the round-trip time implied variations of over an order of magnitude. Comparison of experiments with computational simulations revealed that both methods were strongly affected by experimental artifacts inherent to force spectroscopy measurements, which obscured the intrinsic position-dependence of D. Lastly, we applied Kramers's theory to the kinetics of hairpins with energy barriers located at different positions along the hairpin stem, as a crude probe of D at different stem positions, and we found that D did not vary much as the barrier was moved along the reaction coordinate. This work underlines the difficulties faced when trying to deduce position-dependent diffusion coefficients from experimental folding trajectories.

Prion and prion-like diseases involve the propagation of misfolded protein conformers. Small-molecule pharmacological chaperones can inhibit propagated misfolding, but how they interact with disease-related proteins to prevent misfolding is often unclear. We investigated how pentosan polysulfate (PPS), a polyanion with antiprion activity in vitro and in vivo, interacts with mammalian prion protein (PrP) to alter its folding. Calorimetry showed that PPS binds two sites on natively folded PrP, but one PPS molecule can bind multiple PrP molecules. Force spectroscopy measurements of single PrP molecules showed PPS stabilizes not only the native fold of PrP but also many different partially folded intermediates that are not observed in the absence of PPS. PPS also bound tightly to unfolded segments of PrP, delaying refolding. These observations imply that PPS can act through multiple possible modes, inhibiting misfolding not only by stabilizing the native fold or sequestering natively folded PrP into aggregates, as proposed previously, but also by binding to partially or fully unfolded states that play key roles in mediating misfolding. These results underline the likely importance of unfolded states as critical intermediates on the prion conversion pathway.

No abstract

Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate‐rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form β‐sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern‐day susceptibilities. Ancestral bird PrP could not be seeded with modern‐day prions, just as modern‐day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease.