Biology subjects

Woodside, M. T.

Publications and source records attributed to Woodside, M. T..

11 recordsLinked to original sources

bioRxiv2026

Quantifying the binding affinity of a pharmacological chaperone to transient unfolded states of a normally folded protein

Binding of ligands to partially or fully unfolded proteins can play a key role in the mechanism of cellular and pharmacological chaperones, facilitating proper folding. However, it is challenging to quantify the binding affinity of ligands for unfolded states in a protein that is normally folded, as the methods standardly used to destabilize the native fold also affect ligand binding. We used single-molecule force spectroscopy to unfold single protein molecules without altering solution condi-tions and observe interactions of a ligand with unfolded states. Focusing on pentosan polysulfate (PPS), an anti-prion pharmacological chaperone previously shown to interact with both the native and partially or fully unfolded states of the prion protein, we measured the concentration-dependent effects of PPS binding on the conformational dynamics of bank vole prion protein (BvPrP) molecules held in optical tweezers. We found that PPS stabilized certain partially unfolded intermediate states of BvPrP as well as the fully unfolded state. Strikingly, the tendency to bind unfolded states instead of the folded state increased as the PPS concentration was reduced, implying a higher affinity to unfolded states. From the relative amount of binding to unfolded versus folded states, we estimated that PPS bound roughly 100-fold more tightly to unfolded states than to the native state of PrP. These results reinforce the likely importance of unfolded states in prion misfolding and propagation. More generally, they show how binding affinity to transient, unstable states can be estimated.

biophysics↗

bioRxiv2025

Potent broad-spectrum anti-coronaviral frameshift inhibitors from virtual screen of RNA binding

Coronavirus genomes contain an RNA pseudoknot that directs -1 programmed ribosomal frame-shifting (-1 PRF) to control expression of viral proteins crucial for replication. Ligands that inhibit -1 PRF can thus attenuate viral propagation and have potential as drugs for limiting corona-virus infections. To search for novel small-molecule frameshift inhibitors with anticoronaviral activity, we computationally screened over 14 million compounds for binding to the SARS-CoV-2 pseudoknot, followed by experimental validation of the top hits for inhibition of -1 PRF and viral replication. We identified multiple potent -1 PRF inhibitors, effective at nM concentrations, some of which significantly suppressed SARS-CoV-2 replication in cell culture. Several compounds also inhibited -1 PRF in multiple representative bat coronaviruses, indicating broad-spectrum activity. These results showcase the promise of viral RNA structures like frameshift-stimulatory pseudoknots as targets for broad-spectrum antiviral drugs.

biophysics↗

bioRxiv2025

Deciphering the mechanism of protein aggregation and effects of inhibitors using single-molecule mass photometry

Aggregation of misfolded proteins is a prominent feature of many diseases and hence an attractive drug target. However, the small oligomers that are critical early species in the aggregation cascade are difficult to monitor directly owing to their heterogeneity and transience, complicating efforts to define aggregation mechanisms and target oligomers therapeutically. Here, we observe changes in oligomer populations directly using single-molecule mass photometry (SMMP). Studying the pathogenic P301L mutant of tau protein linked to frontotemporal dementia, we globally fit the growth/decay kinetics for every oligomer population observed by SMMP to microscopic models of aggregation. A simple extension to the best-fit model also accounts for amyloid fibril kinetics, as monitored by Thioflavin T fluorescence, providing the first quantitative model of aggregation kinetics across all stages of the cascade based on direct observation. Crucially, we find that models fitting amyloid kinetics alone fail to capture oligomer behavior, implying that--contrary to standard practice--amyloid kinetics cannot be relied on to deduce aggregation mechanisms. Furthermore, there is no single rate-limiting nucleation step preceding rapid growth, as generally assumed, suggesting that standard models of aggregation are overly simplistic. Repeating the analysis in the presence of aggregation inhibitors allows identification of the discrete steps in the cascade affected by the inhibitors. This work presents a powerful new approach for defining protein aggregation mechanisms and the mechanism of action of inhibitors, with applications to understanding many diseases and developing novel therapeutics.

biophysics↗

bioRxiv2024

Different folding mechanisms in prion proteins from mammals with different disease susceptibility observed at the single-molecule level

Misfolding of the protein PrP causes prion diseases in mammals. Disease susceptibility varies widely among species, despite PrP sequences differing by only a few amino acids. How these differences alter PrP folding and misfolding remains unclear. We compared the folding dynamics of single PrP molecules from three species with different disease susceptibility: dogs (immune), hamsters (susceptible), and bank voles (extremely susceptible). Measurements with optical tweezers revealed important differences between the folding cooperativity, pathways, energy barriers, and kinetics of these proteins. In contrast to the two-state folding of hamster PrP, dog PrP always folded through multiple intermediates. However, both featured rapid native folding, homogeneous energy barriers, and no readily observable misfolding. Bank vole PrP also folded via intermediates, but more slowly and via inhomogeneous barriers. Most notably, it formed several metastable misfolded states starting from the unfolded state. Analyzing the sequence of intermediates seen in pulling curves, we found significant differences in the folding pathways for dog and bank vole PrP, implying that sequence mutations altered energy barriers so as to redirect folding pathways. These results show that subtle differences in PrP sequence between species produce profound changes in folding behavior, providing insight into the factors underlying misfolding propensity.

biophysics↗

bioRxiv2022

Quantifying oligomer populations in real time during protein aggregation using single-molecule mass photometry

Protein aggregation is a hallmark of many neurodegenerative diseases. The early stages of the aggregation cascade are crucial because small oligomers are thought to be key neurotoxic species, but they are difficult to study because they feature heterogeneous mixtures of transient states. We show how the populations of different oligomers can be tracked as they evolve over time during aggregation by using single-molecule mass photometry to measure individually the masses of the oligomers present in solution. Applying the approach to tau protein, whose aggregates are linked to diseases including Alzheimers and frontotemporal dementia, we found that tau existed in an equilibrium between monomers, dimers, and trimers before aggregation was triggered. Once aggregation commenced, the monomer population dropped continuously, paired first with a rise in the population of the smallest oligomers and then a steep drop as the protein was incorporated into larger oligomers and fibrils. Fitting these populations to kinetic models allowed different models of aggregation to be tested, identifying the most likely mechanism and quantifying the microscopic rates for each step in the mechanism. This work demonstrates a powerful new approach for characterizing previously inaccessible regimes in protein aggregation and building quantitative mechanistic models.

biophysics↗

bioRxiv2021

Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted 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 a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

biochemistry↗

bioRxiv2020

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frame-shifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

biophysics↗

bioRxiv2020

Conformational Shannon entropy of mRNA structures from force spectroscopy measurements predicts the efficiency of -1 programmed ribosomal frameshift stimulation

-1 Programmed ribosomal frameshifting (-1 PRF) is stimulated by structures in mRNA, but the factors determining -1 PRF efficiency are unclear. We show that -1 PRF efficiency varies directly with the conformational heterogeneity of the stimulatory structure, quantified as the Shannon entropy of the state occupancy, for a panel of stimulatory structures with efficiency ranging from 2-80%. The correlation is force-dependent and vanishes at forces above those applied by the ribosome. This work supports the hypothesis that heterogeneous conformational dynamics are a key factor in stimulating -1 PRF.

biophysics↗

bioRxiv2020

Mechanically stable knot formed by strand threading in Zika virus RNA confers RNase resistance

Exoribonuclease-resistant RNAs (xrRNAs) from viruses prevent digestion by host exoribonucleases, creating sub-genomic viral RNAs that can enhance infection and pathogenicity. Novel knotted structures in xrRNAs are proposed to act as mechanical road-blocks to RNases. Studying an xrRNA from Zika virus with optical tweezers, we found that it was the most mechanically stable RNA structure yet observed. The knot folded by threading the 5′ end into the cleft of a Mg2+-coordinated three-helix junction before pseudoknot interactions closed a ring around it. Both the threading and pseudoknot were required to generate the extremely force-resistant knot, whose formation correlated directly with RNase resistance both in the wild-type xrRNA and a low-resistance mutant. This work clarifies the folding and mechanism of action of an important new class of RNA.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Anti-frameshifting ligand active against SARS coronavirus-2 is resistant to natural mutations of the frameshift-stimulatory pseudoknot

The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses −1 programmed ribosomal frameshifting (−1 PRF) to control the expression levels of key viral proteins. Because modulating −1 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates −1 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect −1 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the −1 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of −1 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress −1 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal −1 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of −1 PRF.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses -1 programmed ribosomal frameshifting (-1 PRF) to control the relative expression of viral proteins. As modulating -1 PRF can inhibit viral replication, the RNA pseudoknot stimulating -1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by s-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting -1 PRF in SARS-CoV-2.

biophysics↗