Alternative Folding Research Articles

Contact-Map-Driven Exploration of Heterogeneous Protein-Folding Paths.

We have recently shown how physically realizable protein-folding pathways can be generated using directed walks in the space of inter-residue contact-maps; combined with a back-transformation to move from protein contact-maps to Cartesian coordinates, we have demonstrated how this approach can generate protein-folding trajectory ensembles without recourse to molecular dynamics. In this article, we demonstrate that this framework can be used to study a challenging protein-folding problem that is known to exhibit two different folding paths which were previously identified through molecular dynamics simulation at several different temperatures. From the viewpoint of protein-folding mechanism prediction, this particular problem is extremely challenging to address, specifically involving folding to an identical nontrivial compact native structure along distinct pathways defined by heterogeneous secondary structural elements. Here, we show how our previously reported contact-map-based protein-folding strategy can be significantly enhanced to enable accurate and robust prediction of heterogeneous folding paths by (i) introducing a novel topologically informed metric for comparing two protein contact maps, (ii) reformulating our graph-represented folding path generation, and (iii) introducing a new and more reliable structural back-mapping algorithm. These changes improve the reliability of generating structurally sound folding intermediates and dramatically decrease the number of physically irrelevant folding intermediates generated by our previous simulation strategy. Most importantly, we demonstrate how our enhanced folding algorithm can successfully identify the alternative folding mechanisms of a multifolding-pathway protein, in line with direct molecular dynamics simulations.

An intricate balancing act: Upstream and downstream frameshift co-regulatory elements.

Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against Covid-19. During ribosomal translation, a fraction of elongating ribosomes slips by one base in the 5' direction and enters a new reading frame for viral protein synthesis. Any interference with this process profoundly affects viral replication and propagation. For Covid-19, two RNA sites associated with ribosomal frameshifting for SARS-CoV-2 are positioned on the 5' and 3' of the frameshifting residues. Although much attention has been on the 3' frameshift element (FSE), the 5' stem-loop (attenuator hairpin, AH) can play a role. The formation of AH has been suggested to occur as refolding of the 3' RNA structure is triggered by ribosomal unwinding. However, the attenuation activity and the relationship between the two regions are unknown. To gain more insight into these two related viral RNAs and to further enrich our understanding of ribosomal frameshifting for SARS-CoV-2, we explore the RNA folding of both 5' and 3' regions associated with frameshifting. Using our graph-theory-based modeling tools to represent RNA secondary structures, "RAG" (RNA- As-Graphs), and conformational landscapes to analyze length-dependent conformational distributions, we show that AH coexists with the 3-stem pseudoknot of the 3' FSE (graph 3_6 in our dual graph notation) and alternative pseudoknot (graph 3_3) but less likely with other 3' FSE alternative folds (such as 3-way junction 3_5). This is because an alternative length-dependent Stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. In addition, we design four mutants for long lengths that stabilize or disrupt AH, AS1 or FSE pseudoknot to illustrate the deduced AH/AS1 roles and favor the 3_5, 3_6 or stem-loop. These mutants further show how a strengthened pseudoknot can result from a weakened AS1, while a dominant stem-loop occurs with a strengthened AS1. These structural and mutational insights into both ends of the FSE in SARS-CoV-2 advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting a sequence of length-dependent folds, which in turn define potential therapeutic intervention techniques involving both elements. Our work also highlights the complexity of viral landscapes with length-dependent folds, and challenges in analyzing these multiple conformations.

Measuring prion propagation in single bacteria elucidates a mechanism of loss

Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies in mammals, but prions can also constitute nontoxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes. In a fraction of the population, cells had multiple small visible aggregates and lost the prion through random partitioning of aggregates to one of the two daughter cells at division. In the other subpopulation, cells had a stable large aggregate localized to the pole; upon division the mother cell retained this polar aggregate and a daughter cell was generated that contained small aggregates. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation.

Evolutionary selection of proteins with two folds

Although most globular proteins fold into a single stable structure, an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli. State-of-the-art algorithms predict that these fold-switching proteins adopt only one stable structure, missing their functionally critical alternative folds. Why these algorithms predict a single fold is unclear, but all of them infer protein structure from coevolved amino acid pairs. Here, we hypothesize that coevolutionary signatures are being missed. Suspecting that single-fold variants could be masking these signatures, we developed an approach, called Alternative Contact Enhancement (ACE), to search both highly diverse protein superfamilies–composed of single-fold and fold-switching variants–and protein subfamilies with more fold-switching variants. ACE successfully revealed coevolution of amino acid pairs uniquely corresponding to both conformations of 56/56 fold-switching proteins from distinct families. Then, we used ACE-derived contacts to (1) predict two experimentally consistent conformations of a candidate protein with unsolved structure and (2) develop a blind prediction pipeline for fold-switching proteins. The discovery of widespread dual-fold coevolution indicates that fold-switching sequences have been preserved by natural selection, implying that their functionalities provide evolutionary advantage and paving the way for predictions of diverse protein structures from single sequences.

Molecular docking of monkeypox (mpox) virus proteinase with FDA approved lead molecules

BackgroundMonkeypox virus (mpox) disease is caused by a double-stranded DNA virus from the Poxviridae family. The mpox virus showed structural similarity with smallpox virus disease. The recent outbreak of mpox infection in the rest of African countries causes public health issues of increased pandemic potential. Mpox virus is involved in the viral replication cycle through the biocatalytic reaction of precursor polyproteins cleavage. ObjectivesThe main objective of the study was to analyze the molecular interactions between mpox and FDA-approved drugs. MethodsThe primary and secondary structure of the protein was retrieved and FDA approved drug was screened using AutoDock. The best hit was analyzed and the molecular interactions were studied. Model validation analyzes the peptide, energy of hydrogen bonds, steric conflicts and bond planarity. Z-score was calculated using ProSA-web tool and the score tested the native fold from other alternative folds. ResultsThe confidence level of the submitted amino acids was> 80 % and the maximum confidence score for a single template was 98.2 %. The generated proteinase model was subjected to analyze the distribution of atoms and the using ERRAT server. The overall quality score was 88.535 and this value represents the amino acid percentage with anticipated error value and the value falling below the rejection limit. The Z-score of this study result was within the Z-score range (−4.17) validated for native enzymes. The binding pockets of the enzyme were determined in this study and two binding pockets were predicted using the automatic online tool using the web server. The selected FDA-approved drugs were ordered based on their minimum binding energy to the proteinase. ConclusionsMolecular docking studies revealed the involvement of various hydrophobic interactions between FDA-approved drugs and amino acid residues of monkeypox virus proteinase.

ProSeqAProDB: Prosequence Assisted Protein Database

In early 1990s, several proteins were shown to depend on additional stretches of polypeptide (termed as prosequence/prodomain) for their folding. These regions of the protein were often termed as IMCs (Intra Molecular Chaperones), since they would be cleaved from the mature folded protein eventually. Such proteins were hypothesized to face a kinetic barrier to their folding, which was probably lowered by the prosequences. In last three decades, numerous examples of such proteins have accumulated in literature. Yet, no study has been reported so far attempting to understand the evolutionary differences and similaritess of such proteins. Till date such proteins are continued to be treated as anomalous variants, rather than as representatives of any alternate protein folding strategy. Do such proteins have any distinctive structural facets OR typical biological roles, necessitating an unconventional strategy of protein folding? Do prosequences carry any unique or conserved features that are essential to their function?ProSeqAProDb: ProSequence Assisted Protein Database, (which can be accessed at https://proseqaprodb.mkulab.in) was built as a comprehensive database, to systematically study such proteins along with their pro-sequences. The database currently contains 2140 prosequence assisted proteins (1848 eukaryotic, 255 bacterial, 24 viral and 13 archaeal proteins), from 690 organisms later categorised into 960 families. We envisage that the availability of this curated dataset will enable the researchers worldwide to further their investigation in the origin, importance and evolution of such proteins, leading to better understanding of the protein folding process as a whole.

I-Motif folding intermediates with zero-nucleotide loops are trapped by 2′-fluoroarabinocytidine via F···H and O···H hydrogen bonds

G-quadruplex and i-motif nucleic acid structures are believed to fold through kinetic partitioning mechanisms. Such mechanisms explain the structural heterogeneity of G-quadruplex metastable intermediates which have been extensively reported. On the other hand, i-motif folding is regarded as predictable, and research on alternative i-motif folds is limited. While TC5 normally folds into a stable tetrameric i-motif in solution, we report that 2′-deoxy-2′-fluoroarabinocytidine (araF-C) substitutions can prompt TC5 to form an off-pathway and kinetically-trapped dimeric i-motif, thereby expanding the scope of i-motif folding landscapes. This i-motif is formed by two strands, associated head-to-head, and featuring zero-nucleotide loops which have not been previously observed. Through spectroscopic and computational analyses, we also establish that the dimeric i-motif is stabilized by fluorine and non-fluorine hydrogen bonds, thereby explaining the superlative stability of araF-C modified i-motifs. Comparative experimental findings suggest that the strength of these interactions depends on the flexible sugar pucker adopted by the araF-C residue. Overall, the findings reported here provide a new role for i-motifs in nanotechnology and also pose the question of whether unprecedented i-motif folds may exist in vivo.

Evolutionary selection of proteins with two folds.

Although most globular proteins fold into a single stable structure, an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli. State-of-the-art algorithms predict that these fold-switching proteins assume only one stable structure, missing their functionally critical alternative folds. Why these algorithms predict only one fold is unclear, but all of them infer protein structure from coevolved amino acid pairs. Here, we hypothesize that coevolutionary signatures of the unpredicted conformations of fold-switching proteins are being missed. Coevolutionary analysis methods are typically applied to protein superfamilies, which include sequence variants that both do and do not switch folds. Suspecting that single-fold variants could be masking coevolved amino acid pairs required to fold-switch, we developed an approach to analyze diverse protein superfamilies and protein subfamilies with sequences more similar to the target fold-switching sequences. This approach successfully revealed the coevolution of amino acid pairs uniquely corresponding to both conformations of 58 fold-switching proteins from distinct families. Using a set of coevolved amino acid pairs predicted by our approach, we successfully biased AlphaFold2 to predict two experimentally validated conformations of a candidate protein with an unsolved structure. The discovery of widespread dual-fold coevolution indicates that fold-switching sequences have been preserved by natural selection, suggesting that their functionalities provide evolutionary advantage and paving the way for sequence-based predictions of proteins with multiple folds and functions.

Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function.

The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.

RAFFT: Efficient prediction of RNA folding pathways using the fast Fourier transform.

We propose a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. This heuristic is inspired by the kinetic partitioning mechanism, by which molecules follow alternative folding pathways to their native structure, some much faster than others. Similarly, our algorithm RAFFT starts by generating an ensemble of concurrent folding pathways ending in multiple metastable structures, which is in contrast with traditional thermodynamic approaches that find single structures with minimal free energies. When we constrained the algorithm to predict only 50 structures per sequence, near-native structures were found for RNA molecules of length ≤ 200 nucleotides. Our heuristic has been tested on the coronavirus frameshifting stimulation element (CFSE): an ensemble of 68 distinct structures allowed us to produce complete folding kinetic trajectories, whereas known methods require evaluating millions of sub-optimal structures to achieve this result. Thanks to the fast Fourier transform on which RAFFT (RNA folding Algorithm wih Fast Fourier Transform) is based, these computations are efficient, with complexity [Formula: see text].

Alkaline pH has an unexpected effect on transcriptional pausing during synthesis of the Escherichia coli pH-responsive riboswitch

Riboswitches are 5′-untranslated regions of mRNA that change their conformation in response to ligand binding, allowing post-transcriptional gene regulation. This ligand-based model of riboswitch function has been expanded with the discovery of a “pH-responsive element” (PRE) riboswitch in Escherichia coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome-binding sequence, whereas at alkaline pH, the PRE adopts a translationally active structure. This unique riboswitch does not rely on ligand binding in a traditional sense to modulate its alternative folding outcomes. Rather, pH controls riboswitch folding by two possible modes that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that RNAP pausing is prolonged by alkaline pH at two sites, stimulating PRE folding into the active structure. To date, there has been no rigorous exploration into how pH influences RNAP pausing kinetics during PRE synthesis. To provide that understanding and distinguish between pH acting on RNAP versus RNA, we investigated RNAP pausing kinetics at key sites for PRE folding under different pH conditions. We find that pH influences RNAP pausing but not in the manner proposed previously. Rather, alkaline pH either decreases or has no effect on RNAP pause longevity, suggesting that the modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding. These findings invite the possibility that the RNA itself actively participates in the sensing of pH.

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

Mapping Out Histone Post‐Translational Modifications in a Yeast Prion Model

Prions are proteins with the ability to self‐template into alternative folds, allowing them to operate outside the canonical steps of the central dogma of molecular biology. Interestingly, while these infectious protein species are responsible for causing several diseases in humans and animals alike, they have also been postulated to provide positive cellular outcomes in yeast. Because of proteins propensity to misfold and aggregate, prions could allow for environmental adaptiveness without directly affecting an organism’s genome. However, the exact mechanisms by which these proteins could potentially lead to gene utilization changes are not known, and the extent of their roles in normal cellular biology has not been adequately investigated. Rnq1 and Swi1 are two such yeast prion proteins, each responsible for the propagation of the [PIN+] and [SWI+] prion states, respectively. Due to the ease with which prion states exchange, we hypothesize that they could be connected to particular changes in histone post‐translational modifications (PTMs) to alter the way that genes are expressed, and thus could be the key to understanding how prions impact cellular phenotypes. Through previously published methods, we have begun to map out the changes in histone PTM levels between yeast prion states. Overall, [PIN+] yeast showed increased levels of H3K9ac, H3K18ac, and H3K56ac compared to [pin‐]. Interestingly, all these modifications are correlated with increased gene transcription. In agreement, we find higher levels of total RNA in [PIN] yeast. Furthermore, [SWI+] yeast have shown a decrease in H3K56ac and H3K36me2 levels compared to [swi‐] cells. Decreases in these marks generally represent decreased gene expression, which agree with the decrease in global RNA levels in [SWI+] compared to [swi‐] samples. Furthermore, treatment with 1mM guanidinium hydrochloride (GuHCl) ‐ to shut off or “cure” the prion state in yeast‐ reversed the observed histone PTM level changes. Here, we have effectively shown that the yeast epigenome is directly linked to prion states, further confirming the role of prions as another form of epigenetic modulation.

Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways.

Successful de novo protein design ideally targets specific folding kinetics, stability thermodynamics, and biochemical functionality, and the simultaneous achievement of all these criteria in a single step design is challenging. Protein design is potentially simplified by separating the problem into two steps: (a) an initial design of a protein "scaffold" having appropriate folding kinetics and stability thermodynamics, followed by (b) appropriate functional mutation-possibly involving insertion of a peptide functional "cassette." This stepwise approach can also separate the orthogonal effects of the "stability/function" and "foldability/function" tradeoffs commonly observed in protein design. If the scaffold is a protein architecture having an exact rotational symmetry, then there is the potential for redundant folding nuclei and multiple equivalent sites of functionalization; thereby enabling broader functional adaptation. We describe such a "scaffold" and functional "cassette" design strategy applied to a β-trefoil threefold symmetric architecture and a heparin ligand functionality. The results support the availability of redundant folding nuclei within this symmetric architecture, and also identify a minimal peptide cassette conferring heparin affinity. The results also identify an energy barrier of destabilization that switches the protein folding pathway from monomeric to trimeric, thereby identifying another potential advantage of symmetric protein architecture in de novo design.

Selective and Cooperative Photocycloadditions within Multistranded Aromatic Sheets

A series of aromatic helix-sheet-helix oligoamide foldamers composed of several different photosensitive diazaanthracene units have been designed and synthesized. Molecular objects up to 7 kDa were straightforwardly produced on a 100 mg scale. Nuclear magnetic resonance and crystallographic investigations revealed that helix-sheet-helix architectures can adopt one or two distinct conformations. Sequences composed of an even number of turn units were found to fold in a canonical symmetrical conformation with two helices of identical handedness stacked above and below the sheet segment. Sequences composed of an odd number of turns revealed a coexistence between a canonical fold with helices of opposite handedness and an alternate fold with a twist within the sheet and two helices of identical handedness. The proportions between these species could be manipulated, in some cases quantitatively, being dependent on solvent, temperature, and absolute control of helix handedness. Diazaanthracene units were shown to display distinct reactivity toward [4 + 4] photocycloadditions according to the substituent in position 9. Their organization within the sequences was programmed to allow photoreactions to take place in a specific order. Reaction pathways and kinetics were deciphered and product characterized, demonstrating the possibility to orchestrate successive photoreactions so as to avoid orphan units or to deliberately produce orphan units at precise locations. Strong cooperative effects were observed in which the photoreaction rate was influenced by the presence (or absence) of photoadducts in the structure. Multiple photoreactions within the aromatic sheet eventually lead to structure lengthening and stiffening, locking conformational equilibria. Photoproducts could be thermally reverted.

Insulin Complexation with Cyclodextrins-A Molecular Modeling Approach.

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in β-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted β-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5–7 molecules of hydroxypropyl-β-cyclodextrin or sulfobutylether-β-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope

BackgroundThe microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes.ResultsAs starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions.ConclusionsTau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Catalytic Amyloids as Novel Synthetic Hydrolases.

Amyloids are supramolecular assemblies composed of polypeptides stabilized by an intermolecular beta-sheet core. These misfolded conformations have been traditionally associated with pathological conditions such as Alzheimer’s and Parkinson´s diseases. However, this classical paradigm has changed in the last decade since the discovery that the amyloid state represents a universal alternative fold accessible to virtually any polypeptide chain. Moreover, recent findings have demonstrated that the amyloid fold can serve as catalytic scaffolds, creating new opportunities for the design of novel active bionanomaterials. Here, we review the latest advances in this area, with particular emphasis on the design and development of catalytic amyloids that exhibit hydrolytic activities. To date, three different types of activities have been demonstrated: esterase, phosphoesterase and di-phosphohydrolase. These artificial hydrolases emerge upon the self-assembly of small peptides into amyloids, giving rise to catalytically active surfaces. The highly stable nature of the amyloid fold can provide an attractive alternative for the design of future synthetic hydrolases with diverse applications in the industry, such as the in situ decontamination of xenobiotics.

Metamorphic Protein Folding Encodes Multiple Anti-Candida Mechanisms in XCL1.

Candida species cause serious infections requiring prolonged and sometimes toxic therapy. Antimicrobial proteins, such as chemokines, hold great interest as potential additions to the small number of available antifungal drugs. Metamorphic proteins reversibly switch between multiple different folded structures. XCL1 is a metamorphic, antimicrobial chemokine that interconverts between the conserved chemokine fold (an α–β monomer) and an alternate fold (an all-β dimer). Previous work has shown that human XCL1 kills C. albicans but has not assessed whether one or both XCL1 folds perform this activity. Here, we use structurally locked engineered XCL1 variants and Candida killing assays, adenylate kinase release assays, and propidium iodide uptake assays to demonstrate that both XCL1 folds kill Candida, but they do so via different mechanisms. Our results suggest that the alternate fold kills via membrane disruption, consistent with previous work, and the chemokine fold does not. XCL1 fold-switching thus provides a mechanism to regulate the XCL1 mode of antifungal killing, which could protect surrounding tissue from damage associated with fungal membrane disruption and could allow XCL1 to overcome candidal resistance by switching folds. This work provides inspiration for the future design of switchable, multifunctional antifungal therapeutics.