Biology subjects

Itzhaki, L. S.

Publications and source records attributed to Itzhaki, L. S..

17 recordsLinked to original sources

bioRxiv2026

Programmable DNA-peptide nanostructures for multivalent regulation of intracellular signalling

The Wnt/{beta}-catenin pathway is constitutively active in most colorectal and other cancers. Tankyrase (TNKS) promotes Wnt signalling by PARylating AXIN, a rate-limiting scaffold subunit of the {beta}-catenin destruction complex, and its inhibition is a validated strategy for pathway downregulation. Existing small-molecule TNKS inhibitors that target the catalytic PARP domain suffer from off-target effects across the PARP family. Here, we present an alternative approach that targets the substrate binding domains of TNKS using a short TNKS-binding peptide (TBP) presented multivalently on DNA nanostructures. Such a strategy may be required to effectively disrupt the function of targets such as TNKS, which is known to form high-order assemblies in the cell. We engineered DNA nanostructures with two distinct geometries: a 2D triangle (~100 nm) displaying 27 copies of TBP, and a compact 3D tetrahedron (~10 nm) displaying 2 TBP copies. We show that both nanostructures assemble efficiently, can be functionalised with TBP in high yields, and retain binding to TNKS protein in vitro. DNA nanostructures are efficiently internalised to the cytoplasm by HeLa Kyoto and colorectal cancer cells, with TBP functionalisation enhancing rather than hindering uptake. In HeLa cells, both triangle-TBP and tetrahedron-TBP downregulated Wnt signalling to a similar extent despite an order-of-magnitude difference in TBP copy number, while the same concentration of free TBP had no effect. This finding likely reflects the two functions of the DNA nanostructures - intracellular delivery and multivalent display - whereby the smaller nanostructures more efficiently internalise the TBP ligand but have lower valency. These results establish DNA nanostructures as a modular, tuneable platform for multivalent inhibition of intracellular clustered targets such as TNKS.

biophysics↗

bioRxiv2026

DNA aptamers to interfere with HasA, the hemophore of the heme assimilation system in Pseudomonas aeruginosa, as a potential antimicrobial strategy

Pseudomonas aeruginosa relies on heme acquisition to sustain growth and virulence in the iron-limited environment of the host, particularly in the acidic airways of patients with cystic fibrosis where antibiotic efficacy is markedly reduced. The Has heme assimilation system is initiated by the secreted hemophore HasA, which binds extracellular heme and delivers it to the outer-membrane receptor HasR. During infection, HasA is proteolytically processed, generating a truncated form that constitutes the biologically relevant species. Here, we characterise the structural and functional properties of truncated HasA under disease-associated acidic conditions (pH 6.5) and perform a rationally designed Systemic Evolution of Ligands by EXponential enrichment (SELEX) method to identify DNA aptamers capable of binding this hemophore. Biophysical analyses revealed that truncated HasA is folded, displays a mixed /{beta} secondary structure, and exists as a concentration-dependent mixture of monomers and domain-swapped dimers. Circular dichroism and nano-differential scanning fluorimetry identified two thermal transitions, consistent with the coexistence of apo/holo and monomer/dimer species. The apo protein bound heme with high affinity (KD = 113 nM) and a 1:1 stoichiometry, confirming preservation of its functional binding mechanism. Twelve rounds of SELEX, incorporating target switching and platform switching, yielded a highly enriched aptamer pool dominated by two sequences, HasA_1 and HasA_2. ELONA (Enzyme-Linked Oligonucleotide Assay) assays demonstrated that both aptamers bind specifically to truncated HasA, and biolayer interferometry revealed nanomolar dissociation constants (KD = 0.59 M and 0.32 M, respectively). These aptamers did not bind full-length HasA, BSA, or control sequences, confirming that selection under acidic conditions drove specificity toward the physiologically relevant form of the hemophore. Our findings identify HasA-binding DNA aptamers that retain function in acidic environments where antibiotic potency is compromised, highlighting their potential as molecular tools to disrupt heme acquisition in P. aeruginosa. This work establishes a foundation for developing aptamer-based antimicrobial strategies targeting the Has system, particularly under acidic conditions where conventional antibiotics are compromised.

biophysics↗

bioRxiv2026

A generalisable method for the purification and biophysical characterisation of bacterial membrane receptors

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

biophysics↗

bioRxiv2026

Combinatorial screening of biological degrader modules identifies heterobifunctional molecules that efficiently destroy MYC, leading to rapid tumour cell death in vitro and in vivo

Targeted protein degradation is a potent strategy against intracellular proteins impervious to traditional drugs. We describe a platform reliant on high-throughput cloning and mRNA-based delivery to quickly screen 100s-1000s of modular biological degraders, which we challenge against the high-turnover c-Myc oncoprotein. We uncover critical principles to drive degrader discovery against any target, free from tagging or prior cell line engineering, with direct applications in research and potentially therapy. We demonstrate the feasibility of targeted protein degradation of c-Myc across in vitro cancer models and in vivo xenograft model, uncovering distinct cellular responses between c-Myc degradation and inhibition.

bioengineering↗

bioRxiv2025

Coiled-coil scaffolds for Targeted Protein Degradation

In targeted protein degradation (TPD) the cells quality control machinery is co-opted to degrade proteins of interest. Currently TPD is limited by the availability of small-molecule binders for targets and the many potential human E3 ubiquitin ligases. Consequently, other approaches are needed to tackle new targets and to exploit degradation pathways fully. The natural binding epitopes for E3s and other degradation machineries are often short linear peptide motifs, which offer routes to new biologics-based degraders. Here, targeting the anti-apoptotic BCL-xL and initiation of apoptosis, we show that several protein-degradation pathways--ubiquitination, direct proteasome, autophagy-lysosome recruitment--can be leveraged by integrating target-binding and degradation motifs into single-polypeptide constructs. This exploits versatile and varied de novo coiled-coil scaffolds as adaptable molecular glues by presenting motifs multivalently and in diverse arrangements. The constructs are small ({approx}80 residues) and provide a plug-and-play platform to map and optimise target-degradation space using rational design. We call this Coiled Coils for induced Proximity (CoCoProx) and propose its use for targeted degradation and other induced-proximity reactions.

synthetic biology↗

bioRxiv2025

De novo designed bifunctional proteins for targeted protein degradation

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot-binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.

synthetic biology↗

bioRxiv2025

Small molecule activator of phosphatase PP2A remodels scaffold PR65 structural dynamics to promote holoenzyme assembly

Small molecule activators of protein phosphatase 2A (PP2A), hereafter SMAPs, have attracted substantial interest, for their potential to inhibit cancer cell proliferation by targeting PR65, the scaffold subunit of the PP2A heterotrimer. PR65 is a uniquely flexible and stable molecule composed of 15 tandem HEAT (Huntingtin, Elongation factor 3-PP2A-TOR1) repeats. We characterized the binding sites and interactions of two SMAPs ATUX-8385 and DT-061 with PR65 and evaluated effects on PR65 structural dynamics using docking and molecular dynamics simulations. We initiated SMAP-bound PR65 simulations starting from two binding sites: S1, determined by cryo-electron microscopy for DT-061 bound to PP2A, on the inner helices of the HEAT repeats 2 and 3 (2i and 3i); and S2, predicted by docking of ATUX-8385 onto PR65, on 4i and 5i and outer helices 5o and 6o consistent with footprinting experiments. S2 proved to be a stable site for both SMAPs when initiating the simulations at S2. However, neither DT-061 nor ATUX-8385 demonstrated stable binding to S1. DT-061 rapidly dissociated from S1 to settle instead at a neighboring site S4 overlapping with our previously identified S3 for PR65 in extended form, suggesting that binding to S1 may be a 2-step process: an initial binding to PR65 alone, either to S3/S4 or S2, followed by movement to S3/S4, and then an induced relocation to S1 upon complexation with the regulatory and catalytic subunits. Targeted in silico mutagenesis showed that mutations at S2 and S4 destabilized SMAP binding to the PR65 (subunit). Heterotrimeric PP2A simulations showed that S3 and S4 binding were not persistent upon complexation. Together, these results corroborate our findings. Furthermore, preferentially stabilized a relatively extended PR65 conformation that would accommodate, if not promote, the assembly of the catalytic and regulatory subunits to prompt the activation of the trimeric phosphatase.

biophysics↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2025

Using a stable protein scaffold to display peptides that bind to alpha-synuclein fibrils

Amyloid fibrils are ordered aggregates that are a pathological hallmark of many neurodegenerative disorders including Alzheimers Disease and Parkinsons Disease. The process of amyloid formation involves a complex cascade by which soluble monomeric protein converts to insoluble, ordered aggregates (amyloid fibrils). Although inhibiting the aggregation pathway is a key target for therapeutic development, the heterogeneous collection of aggregation-prone species formed in this process, including oligomers, protofibrils and fibrils, represent other targets for modifying disease pathology. Developing molecules that can bind to amyloid fibrils and potentially disrupt the harmful interactions between the fibrils and the cellular components would be advantageous. Designing peptide modulators for -synuclein aggregation is of great interest, however effective inhibitory peptides are often hydrophobic and hence difficult to handle; therefore, developing strategies to display these peptides in a soluble scaffold would be very beneficial. Here we demonstrate that the ultra-stable consensus-designed tetratricopeptide repeat (CTPR) protein scaffold can be grafted with "KLVFF" derived peptides previously identified to inhibit protein aggregation and interact with amyloid fibrils, to produce proteins that bind along the surface of -synuclein fibrils with micromolar affinity. Given the ability to insert hydrophobic peptides to produce soluble, CTPR-based binders, this method may prove beneficial in screening for peptide modulators of protein aggregation. Short statementConsensus-designed tetratricopeptide repeat (CTPR) proteins can be endowed with additional functionality including specific client recruitment. Here we created stable, soluble CTPR-based binders that recognise -synuclein fibrils with micromolar affinity. The CTPR scaffold provides a facile way to test potential fibril-binding or aggregation-modulating peptides and to probe their mechanisms of action.

biochemistry↗

bioRxiv2024

Development of D-box peptides to inhibit the Anaphase Promoting Complex/Cyclosome

E3 ubiquitin ligases engage their substrates via degrons - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are hard to drug via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the Destruction box (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20- peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/CCdc20 and are more potent than the small molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially also for harnessing APC/C for targeted protein degradation.

biochemistry↗

bioRxiv2024

Tandem-repeat proteins introduce tuneable properties to engineered biomolecular condensates

The cells ability to rapidly partition biomolecules into biomolecular condensates is linked to a diverse range of cellular functions. Understanding how the structural attributes of biomolecular condensates are linked with their biological roles can be facilitated by the development synthetic condensate systems that can be manipulated in a controllable and predictable way. Here, we design and characterise a tuneable synthetic biomolecular condensate platform fusing modular consensus-designed tetratricopeptide repeat (CTPR) proteins to intrinsically-disordered domains. Trends between the CTPR structural attributes and condensate propensity were recapitulated across different experimental conditions and by in silico modelling, demonstrating that the CTPR domain can systematically affect the condensates in a predictable manner. Moreover, we show that incorporating short binding motifs into the CTPR domain results in specific target-protein recruitment into the condensates. Our model system can be rationally designed in a versatile manner to both tune condensate propensity and endow the condensates with new functions.

biophysics↗

bioRxiv2024

Investigating the interactions of the cucumber mosaic virus 2b protein with the viral 1a replicase component and the cellular RNA silencing factor Argonaute 1

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b proteins host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMVs insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b proteins interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b proteins ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

plant biology↗

bioRxiv2024

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of anti-malarial therapeutics.

biophysics↗

bioRxiv2021

Parallel and sequential pathways of molecular recognition of a tandem-repeat protein and its intrinsically disordered binding partner

The Wnt signalling pathway plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein {beta}-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between {beta}-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 [A]2 that spans ten of the twelve ARM repeats of {beta}-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and {beta}-catenin was monophasic and rapid (7.3 {+/-} 0.1 x107 M-1s-1), whereas dissociation was biphasic and slow (5.7 {+/-} 0.4 x10-4 s-1, 15.2 {+/-} 2.8 x10-4 s-1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2-{beta}-catenin complex. We found that mutation of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures has a large effect on the dissociation kinetics, whereas mutation of the labile sub-domain connecting them has negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a "fuzzy" complex involving transient contacts that are not site-specific. Strikingly, two mutations in the N-terminal subdomain that have the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

biophysics↗

bioRxiv2021

Consensus tandem tetratricopeptide repeat proteins are complex superhelical nanosprings

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs) -- superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between folded and unfolded conformations. We rationalise the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the centre towards both termini simultaneously. Strikingly, we also directly observe the proteins superhelical tertiary structure in the force signal. Using protein engineering, crystallography and single-molecule experiments, we show how the superhelical geometry can be altered by carefully placed amino-acid substitutions and examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications. Significance statementRepetition of biological building blocks is crucial to modulating and diversifying structure and function of biomolecules across all organisms. In tandem-repeat proteins, the linear arrangement of small structural motifs leads to the formation of striking supramolecular shapes. Using a combination of single-molecule biophysical techniques and modelling approaches, we dissect the spring-like nature of a designed repeat protein and demonstrate how its shape and mechanics can be manipulated by design. These novel insights into the biomechanical and biochemical characteristics of this protein class give us a methodological basis from which to understand the biological functions of repeat proteins and to exploit them in nanotechnology and biomedicine.

biophysics↗

bioRxiv2019

Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azide-alkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.

biophysics↗

bioRxiv2017

PyFolding: An open-source software package for graphing, analysis and simulation of thermodynamic and kinetic models of protein folding

Abstract]For many years, curve fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large datasets. One field that is relient on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open source, and extensible Python framework for graphing, analysis and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: (i) multi-phase kinetic folding fitted to linked equations, (ii) global fitting of multiple datasets and (iii) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how Pyfolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and amongst research teams.

biochemistry↗