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.

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.

Integration of Control Strategies to Optimize Management of Ambrosia Beetles (Coleoptera: Curculionidae, Scolytinae) and Phytophthora Root Rot (Peronosporales: Peronosporaceae) in Flowering Dogwoods (Cornalaes: Cornaceae) After Simulated Flooding.

Ambrosia beetles (Coleoptera: Curculionidae, Scolytinae) and Phytophthora root rot (Peronosporales: Peronosporaceae) cause significant damage to the ornamental industry in the United States. In this study, mefenoxam (fungicide), permethrin (insecticide), and charcoal + kaolin were used in different combinations with Phytophthora cinnamomi (Rands: Peronosporales: Peronosporaceae) inoculated and noninoculated plants to optimize the management of ambrosia beetles and Phytophthora root rot. Treatment applications were performed in two trials on 1 (mefenoxam, drench), 18 (P. cinnamomi inoculation), or 19 (permethrin, spray) days before instigating flood stress or 2 d after flood stress (charcoal + kaolin, spray), respectively. Flooding was maintained for 21 d. Ambrosia beetle attacks and plant growth data were recorded. Tree roots were rated at study end for disease severity and root samples were plated on PARPH-V8 medium to determine the percentage of pathogen recovery. In both trials, the combination of mefenoxam + permethrin treatment had reduced disease severity and ambrosia beetle attacks compared to the inoculated controls. Permethrin-treated trees had shorter galleries compared to controls in trial 1 and no gallery formation in trial 2. In both trials, no differences were observed among the treatments in numbers of galleries with eggs and adults, but mefenoxam + charcoal + kaolin had significantly fewer galleries with larvae among the noninoculated trees compared with the respective control in trial 1. Overall, treatments containing combinations of mefenoxam + permethrin had reduced disease severity and ambrosia beetle attacks.

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.

Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses.

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.

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.

Association of geographic distribution and birth weight with sociodemographic factors of the maternal and newborn child of hilly and mountain regions of eastern Nepal: a cross-sectional study.

OBJECTIVES: To determine the association of geographic distribution, and birth weight with sociodemographic factors of the maternal and newborn child of hilly region (lower altitude) and mountain region (high altitude) of eastern Nepal as well as the prevalence of low birth weight (LBW) and large for gestational age (LGA) among term singleton deliveries in eastern Nepal. METHODOLOGY: A cross-sectional study was conducted in the district-level hospitals of Dhankuta, Tehrathum, Solukhumbu and Taplejung districts of eastern Nepal of Province 1. Mothers with preterm or post-term delivery, multiple pregnancies, stillbirth/intrauterine fetal death and incomplete records were excluded from the study with only 1386 term pregnancies (37-42 weeks) delivered at the respective facilities between 17 July 2019 and 16 July 2020 were included. The appropriate data were entered in Microsoft Excel 2019 V.16.0 and statistical analysis was performed by using the statistical package for social sciences, IBM SPSS V.29. RESULTS: The low maternal age, Dalit ethnic group, low gravidity, low parity, higher antenatal care (ANC) visits (≥4), incomplete deworming and dT vaccination status, breech deliveries and LBW newborns were significantly attributed to hilly region (lower altitude) (p value <0.05). Similarly, the hilly region, lower and/or no ANC visits and early term gestation had significant negative association with birth weight at the lower quantiles only. Meanwhile, the female newborn had significant and negative association with birth weight distribution at all seven quantiles. The prevalence of the LBW, average for gestational age and LGA newborn child among term singleton deliveries in Eastern Nepal is 6.6%, 85.8% and 7.6%, respectively. CONCLUSIONS: The local organisations should focus on adequate antenatal care visits in mountain region and coverage of dT vaccine and deworming medications in hilly region. Appropriate measures and programmes should be initiated to bring down LBW in hilly region.

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.

Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers.

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

Anti-Frameshifting Ligand Active against SARS Coronavirus-2 Is Resistant to Natural Mutations of the Frameshift-Stimulatory Pseudoknot.

SARS-CoV-2 uses -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins. Because modulating -1 PRF can attenuate the virus, ligands binding to the RNA pseudoknot that stimulates -1 PRF may have therapeutic potential. Mutations in the pseudoknot have occurred during the pandemic, but how they affect -1 PRF efficiency and ligand activity is unknown. Studying a panel of six mutations in key regions of the pseudoknot, we found that most did not change -1 PRF levels, even when base-pairing was disrupted, but one led to a striking 3-fold decrease, suggesting SARS-CoV-2 may be less sensitive to -1 PRF modulation than expected. Examining the effects of a small-molecule -1 PRF inhibitor active against SARS-CoV-2, it had a similar effect on all mutants tested, regardless of basal -1 PRF efficiency, indicating that anti-frameshifting activity can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of -1 PRF.

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.

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

17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had 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 provide promising lead compounds to counter the current pandemic.

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.

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.

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.

Testing Kinetic Identities Involving Transition-Path Properties Using Single-Molecule Folding Trajectories.

Recent advances in single-molecule assays have allowed individual transition paths during the folding of single molecules to be observed directly. We used the transition paths of DNA hairpins having different sequences, measured with high-resolution optical tweezers, to test theoretical relations between the properties of the transition paths and the folding kinetics. We showed that folding and unfolding rates were related to the average transition-path times, as expected from theory, for all hairpins studied. We also found that the probability distribution of transition-path occupancies agreed with the profile of the average velocity along the transition paths for each of the hairpins, as expected theoretically. Finally, we used the latter result to show that the committor probability recovered from the velocity profile matches the committor measured empirically. These results validate the proposed kinetic identities.

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.

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.

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.

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

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.