Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins.

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy-landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

Nonlinear effects in optical trapping of titanium dioxide and diamond nanoparticles.

Optical trapping in biophysics typically uses micron-scale beads made of materials like polystyrene or glass to probe the target of interest. Using smaller beads made of higher-index materials could increase the time resolution of these measurements. We characterized the trapping of nanoscale beads made of diamond and titanium dioxide (TiO2) in a single-beam gradient trap. Calculating theoretical expectations for the trapping stiffness of these beads, we found good agreement with measured values. Trap stiffness was significantly higher for TiO2 beads, owing to notable enhancement from nonlinear optical effects, not previously observed for continuous-wave trapping. Trap stiffness was over 6-fold higher for TiO2 beads than polystyrene beads of similar size at 70 mW laser power. These results suggest that diamond and TiO2 nanobeads can be used to improve time resolution in optical tweezers measurements.

Acibenzolar-S-methyl induces resistance against ambrosia beetle attacks in dogwoods exposed to simulated flood stress.

Xylosandrus spp. ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are important wood-boring pests of nursery trees weakened by abiotic and biotic stressors. Acibenzolar-S-methyl (ASM), a plant defense elicitor, was tested for inhibiting Xylosandrus spp. tunneling (i.e., attacks) into flood-stressed flowering dogwoods (Cornus florida L. (Cornales: Cornaceae)). Container-grown dogwoods were treated with ASM substrate drench + flooding, ASM foliar spray + flooding, ASM drench + no flooding, ASM foliar + no flooding, no ASM + flooding, or no ASM + no flooding at 3 days before flood stress in a completely randomized design under field conditions. Trees were flooded for 14 days and then drained and watered as needed. Attacks were counted every 2 days for 28 days. Plant tissue samples were collected at 7 and 14 days after flooding to determine ethanol content using solid-phase microextraction-gas chromatography-mass spectrometry. Trees were dissected to determine gallery formation and depth, fungal colonization, and the presence of eggs, larvae, and adults. The highest number of Xylosandrus beetle species attacks were recorded from plants exposed to no ASM + flooding, but attacks were reduced in ASM treated trees (drench or foliar) + flooding. Trees treated with drenches had fewer attacks than foliar sprays. Plants assigned to no flood had the fewest beetle attacks. Moreover, ASM reduced Xylosandrus spp. gallery formation and depth, fungal colonization, and presence of eggs, larvae, and adults. All flooded trees produced ethanol. In conclusion, ASM induced a plant defense response to Xylosandrus spp. tunneling in dogwoods under flood stress conditions.

Measuring the average shape of transition paths during the folding of a single biological molecule.

Transition paths represent the parts of a reaction where the energy barrier separating products and reactants is crossed. They are essential to understanding reaction mechanisms, yet many of their properties remain unstudied. Here, we report measurements of the average shape of transition paths, studying the folding of DNA hairpins as a model system for folding reactions. Individual transition paths were detected in the folding trajectories of hairpins with different sequences held under tension in optical tweezers, and path shapes were computed by averaging all transitions in the time domain, 〈t(x)〉, or by averaging transitions of a given duration in the extension domain, 〈x(t|τ)〉 τ Whereas 〈t(x)〉 was close to straight, with only a subtle curvature, 〈x(t|τ)〉 τ had more pronounced curvature that fit well to theoretical expectations for the dominant transition path, returning diffusion coefficients similar to values obtained previously from independent methods. Simulations suggested that 〈t(x)〉 provided a less reliable representation of the path shape than 〈x(t|τ)〉 τ , because it was far more sensitive to the effects of coupling the molecule to the experimental force probe. Intriguingly, the path shape variance was larger for some hairpins than others, indicating sequence-dependent changes in the diversity of transition paths reflective of differences in the character of the energy barriers, such as the width of the barrier saddle-point or the presence of parallel paths through multiple barriers between the folded and unfolded states. These studies of average path shapes point the way forward for probing the rich information contained in path shape fluctuations.

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure.

Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates -1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during -1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of -1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.

Efficacy and Timing of Application of Fungicides, Biofungicides, Host-Plant Defense Inducers, and Fertilizer to Control Phytophthora Root Rot of Flowering Dogwood in Simulated Flooding Conditions in Container Production.

Phytophthora root rot, caused by Phytophthora cinnamomi Rands, is one of the major diseases of flowering dogwood (Cornus florida L.). The severity of root rot disease increases when the plants are exposed to flooding conditions. A study was conducted to determine the efficacy and timing of application of different fungicides, biofungicides, host-plant defense inducers, and fertilizer to manage Phytophthora root rot in month-old seedlings in simulated flooding events for 1, 3, and 7 days. Preventative treatments were drench applied 3 weeks and 1 week before flooding whereas curative treatments were applied 24 h after flooding. Dogwood seedlings were inoculated with P. cinnamomi 3 days before the flooding. Plant height and width were recorded at the beginning and end of the study. At the end of the study, plant total weight and root weight were recorded and disease severity in the root was assessed using a scale of 0 to 100%. Root samples were plated using PARPH-V8 medium to determine the percent recovery of the pathogen. Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX, as preventative and curative applications, were able to suppress the disease severity compared with the inoculated control in all flooding durations. All treatments, with the exception of Stargus as a preventative application 3 weeks before flooding and Orkestra Intrinsic as a curative application, were able to suppress the disease severity compared with the inoculated control for a 1-day flooding event. Aliette and ON-Gard were effective in the first trial when applied preventatively at both 1 week and 3 weeks before flooding but not in the second trial. Signature Xtra was effective as a preventative application but not as a curative application. Interface was effective as a curative application but not as a preventative application. The findings of this study will help nursery growers to understand the performance of fungicides, biofungicides, host-plant defense inducers, and fertilizer at different time intervals and repeated applications to manage Phytophthora root rot in flooding conditions.

Management of Phytophthora cinnamomi Using Fungicides and Host Plant Defense Inducers Under Drought Conditions: A Case Study of Flowering Dogwood.

Phytophthora cinnamomi is considered one of the most destructive pathogens of ornamental crops. Different fungicides and host plant defense inducers were tested for their efficacy in managing Phytophthora root rot in drought conditions. In this study, the drought conditions were maintained by evaluating the moisture-holding capacity of the pine bark in a 10.2-cm nursery container. Four controls and nine different treatments were used in two trials for this greenhouse study. All treatments were drench-applied as a preventative or curative treatment. Seedlings were artificially inoculated with P. cinnamomi. Regular irrigation was carried out using overhead irrigation for 1 month after inoculation. Irrigation was regulated by drip irrigation after the first month. A moisture level of 15% to 18% of total moisture-holding capacity was maintained using the gravimetric method throughout the drought period. Physiological parameters of the seedlings were recorded a week after seedlings were drought stressed. In both trials of preventative and curative treatments, all treatments were able to suppress the disease significantly except Orkestra Intrinsic. Orkestra Intrinsic had a disease severity statistically similar to the inoculated and stressed control in trial 1 of the curative treatment. Net photosynthesis, stomatal conductance, and leaf moisture potential were significantly greater in seedlings treated with Subdue MAXX, Signature Xtra, and Empress Intrinsic in both trials of preventative and curative treatments. Effective quantum yield of Photosystem II was significantly lower in the inoculated stressed control in both trials of preventative and curative treatments. Net chlorophyll content through the SPAD meter showed higher values for Subdue MAXX treated seedlings compared with the noninoculated nonstressed controls in trial 1 as both a curative and preventative application. In trial 2, Subdue MAXX and Signature Xtra were the best curative treatments, whereas Empress Intrinsic, Interface, and Subdue MAXX were the best preventative treatments for higher chlorophyll content. This case study will help growers perform successful management of Phytophthora root rot in woody ornamental crops during drought or water deficit conditions.

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2).

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation.

The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0 to 100%, we determined both the average time to cross the transition paths, τtp, and the distribution of individual transit times, PTP(t). We then estimated D from both τtp and PTP(t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. τtp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base pairs than for those with only G:C base pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0 to 100%. These results reveal that G:C base pairs form faster than A:T base pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding.

Measuring the Local Velocity along Transition Paths during the Folding of Single Biological Molecules.

Transition paths are the most interesting part of folding reactions but remain little studied. We measured the local velocity along transition paths in DNA hairpin folding using optical tweezers. The velocity distribution agreed well with diffusive theories, yielding the diffusion coefficient. We used the average velocity to calculate the transmission factor in transition-state theory (TST), finding observed rates that were ∼10^{5}-fold slower than predicted by TST. This work quantifies the importance of barrier recrossing events and highlights the effectiveness of the diffusive model of folding.

Transition-path properties for folding reactions in the limit of small barriers.

Transition paths are of great interest because they encapsulate information about the mechanisms of barrier-crossing reactions. Analysis of experiments measuring biomolecular folding reactions has relied on expressions for properties of transition paths such as transition-path times and velocities that hold in the limit of large harmonic barriers, but real molecules often have relatively small barriers. Recent theoretical work presented more general expressions for transition-path properties. Here we extend this work, deriving expressions from the general case that can be applied to small harmonic barriers. We first compared the performance of small-barrier, large-barrier, and general solutions when applied to simulated transitions, focusing on improvements in estimates of the diffusion coefficient determined from transition times and velocities. We then applied these expressions to experimental data from force spectroscopy measurements of DNA hairpins. We found that the low-barrier approximation and exact solution reduced or resolved the small but systematic inconsistencies that had arisen from assuming large harmonic barriers, demonstrating the practical utility of the new equations for analyzing experimental data.

Quantifying Instrumental Artifacts in Folding Kinetics Measured by Single-Molecule Force Spectroscopy.

Force spectroscopy is commonly used to measure the kinetics of processes occurring in single biological molecules. These measurements involve attaching the molecule of interest to micron-sized or larger force probes via compliant linkers. Recent theoretical work has described how the properties of the probes and linkers can alter the observed kinetics from the intrinsic behavior of the molecule in isolation. We applied this theory to estimate the errors in measurements of folding made using optical tweezers. Errors in the folding rates arising from instrument artifacts were only ∼20% for constant-force measurements of DNA hairpins with typical choices of linker length and probe size. Measurements of transition paths using a constant trap position at high trap stiffness were also found to be in the low-artifact limit. These results indicate that typical optical trap measurements of kinetics reflect the dynamics of the molecule fairly well, and suggest practical limitations on experimental design to ensure reliable kinetic measurements.

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates.

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.

Direct observation of multiple misfolding pathways in a single prion protein molecule.

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 high-resolution 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 off-pathway 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.

Single-molecule force spectroscopy of rapidly fluctuating, marginally stable structures in the intrinsically disordered protein α-synuclein.

Intrinsically disordered proteins form transient, fluctuating structures that are difficult to observe directly. We used optical tweezers to apply force to single α-synuclein molecules and measure their extension, characterizing the resulting conformational transitions. Force-extension curves revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few kBT, but the diffusion was slow, revealing a landscape that is flat but rough.

Elasticity, viscosity, and orientational fluctuations of a lyotropic chromonic nematic liquid crystal disodium cromoglycate.

Using dynamic light scattering, we study orientational fluctuation modes in the nematic phase of a self-assembled lyotropic chromonic liquid crystal (LCLC) disodium cromoglycate and measure the Frank elastic moduli and viscosity coefficients. The elastic moduli of splay (K1) and bend (K3) are in the order of 10 pN while the twist modulus (K2) is an order of magnitude smaller. The splay constant K1 and the ratio K1/K3 both increase substantially as the temperature T decreases, which we attribute to the elongation of the chromonic aggregates at lower temperatures. The bend viscosity is comparable to that of thermotropic liquid crystals, while the splay and twist viscosities are several orders of magnitude larger. The temperature dependence of bend viscosity is weak. The splay and twist viscosities change exponentially with the temperature. In addition to the director modes, the fluctuation spectrum reveals an additional mode that is attributed to diffusion of structural defects in the column-like aggregates.

Single-molecule assays for investigating protein misfolding and aggregation.

Protein misfolding and aggregation are relevant to many fields. Recently, their investigation has experienced a revival as a central topic in the research of numerous human diseases, including Parkinson's and Alzheimer's. Much has been learned from ensemble biochemical approaches, but the inherently heterogeneous nature of the underlying processes has obscured many important details. Single-molecule techniques offer unique capabilities to study heterogeneous systems, while providing high temporal and structural resolution to characterize them. In this Perspective, we give an overview of the single-molecule assays that have been applied to protein misfolding and aggregation, which are mainly based on fluorescence and force spectroscopy. We describe some of the technical challenges involved in studying aggregation at the single-molecule level and discuss what has been learned about aggregation mechanisms from the different approaches.

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism.

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.

Transition path times for nucleic Acid folding determined from energy-landscape analysis of single-molecule trajectories.

The duration of structural transitions in biopolymers is only a fraction of the time spent searching diffusively over the configurational energy landscape. We found the transition time, τ(TP), and the diffusion constant, D, for DNA and RNA folding using energy landscapes obtained from single-molecule trajectories under tension in optical traps. DNA hairpins, RNA pseudoknots, and a riboswitch all had τ(TP)~10 µs and D~10(-13-14) m(2)/s, despite widely differing unfolding rates. These results show how energy-landscape analysis can be harnessed to characterize brief but critical events during folding reactions.