Biology subjects

Roy, R.

Publications and source records attributed to Roy, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Multiomic Single Cell Sequencing Identifies Stemlike Nature of Mixed Phenotype Acute Leukemia and Provides Novel Risk Stratification

Mixed phenotype acute leukemia (MPAL) is a leukemia whose biologic drivers are poorly understood, therapeutic strategy remains unclear, and prognosis is poor. We performed multiomic single cell (SC) profiling of 14 newly diagnosed adult MPAL patients to characterize the immunophenotypic, genetic, and transcriptional landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. However, progressive acquisition of mutations is associated with increased expression of immunophenotypic markers of immaturity. Using SC transcriptional profiling, we find that MPAL blasts express a stem cell-like transcriptional profile distinct from other acute leukemias and indicative of high differentiation potential. Further, patients with the highest differentiation potential demonstrated inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in this cohort, is applicable to bulk RNA sequencing data and was predictive of survival in an independent patient cohort, suggesting utility for clinical risk stratification.

cancer biology↗

bioRxiv2023

Cholesterol catalyzes unfolding in membrane inserted motifs of the pore forming protein cytolysin A

Plasma membrane induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize and form transmembrane pores. Unregulated pore formation causes ion imbalance leading to cell death and infection. Determining the free energy landscape of these membrane driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the -PFT, cytolysin A expressed by E. coli is less understood. In a recent free energy computation, we have shown that the {beta}-tongue, a critical membrane inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state.1 To understand the molecular role played by cholesterol, we have carried out string method based computations in membranes devoid of cholesterol which reveals an increase of[~] 30 times in the free energy barrier for the loss of {beta}-sheet secondary structure when compared with membranes containing cholesterol. Specifically the tyrosine-cholesterol interaction was found to be critical to stabilizing the unfolded intermediate. In the absence of cholesterol the membrane was found to undergo large curvature deformations in both leaflets of the membrane accompanied by bilayer thinning. Our study with the -toxin, ClyA illustrates that cholesterol is critical to catalyzing and stabilizing the unfolded state of the {beta}-tongue in the membrane, opening up fresh insights into cholesterol assisted unfolding of membrane proteins. SignificanceCholesterol, an integral part of mammalian cell membranes, is necessary for activity of pathogenic toxins. Our understanding of the thermodynamic and molecular underpinnings of cholesterol-protein interactions during different stages of toxin activity is unclear. Using path based all atom molecular dynamics simulations, we illustrate lowered free energy barriers and enhanced stability of the membrane unfolded intermediate of an -pore forming toxin (PFT) ClyA providing insights into the increased pore formation kinetics with cholesterol. Thus, membrane cholesterol generally believed to play a passive receptor function for PFT activity is involved in a more complex regulatory role in assisting secondary structure transitions critical to PFT lytic activity. Our findings could aid in drug development strategies for mitigating PFT mediated bacterial infections.

biophysics↗

bioRxiv2023

Torch-eCpG: A fast and scalable eQTM mapper for thousands of molecular phenotypes with graphical processing units

BackgroundGene expression may be regulated by the DNA methylation of regulatory elements in cis, distal, and trans regions. One method to evaluate the relationship between DNA methylation and gene expression is the mapping of expression quantitative trait methylation (eQTM) loci (also called expression associated CpG loci, eCpG). However, no open-source tools are available to provide eQTM mapping. In addition, eQTM mapping can involve a large number of comparisons which may prevent the analyses due to limitations of computational resources. Here, we describe Torch-eCpG, an open-source tool to perform eQTM mapping that includes an optimized implementation that can use the graphical processing unit (GPU) to reduce runtime. ResultsWe demonstrate the analyses using the tool are reproducible, up to 18x faster using the GPU, and scale linearly with increasing methylation loci. ConclusionsTorch-eCpG is a fast, reliable, and scalable tool to perform eQTM mapping. Source code for Torch-eCpG is available at https://github.com/kordk/torch-ecpg.

bioinformatics↗

bioRxiv2023

Automatic Detection of Cell-cycle Stages using Recurrent Neural Networks

Mitosis is the process by which eukaryotic cells divide to produce two similar daughter cells with identical genetic material. Research into the process of mitosis is therefore of critical importance both for the basic understanding of cell biology and for the clinical approach to manifold pathologies resulting from its malfunctioning, including cancer. In this paper, we propose an approach to study mitotic progression automatically using deep learning. We used neural networks to predict different mitosis stages. We extracted video sequences of cells undergoing division and trained a Recurrent Neural Network (RNN) to extract image features. The use of RNN enabled better extraction of features. The RNN-based approach gave better performance compared to classifier based feature extraction methods which do not use time information. Evaluation of precision, recall, and F-score indicates the superiority of the proposed model compared to the baseline. To study the loss in performance due to confusion between adjacent classes, we plotted the confusion matrix as well, to quantify the amount of misclassification. In addition, we visualized the feature space to understand why RNNs are better at classifying the mitosis stages than other classifier models, which indicated the formation of strong clusters for the different classes, clearly confirming the advantage of the proposed RNN-based approach.

bioinformatics↗

bioRxiv2023

Transcription elongation defects link oncogenic splicing factor mutations to targetable alterations in chromatin landscape

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two splicing factors commonly mutated in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy. HIGHLIGHTS- Oncogenic mutations in SF3B1 and U2AF1 cause a gene body RNAPII transcription elongation defect - The elongation defect is linked to impaired assembly of early spliceosome complexes and leads to replication stress and changes to chromatin landscape - RNAPII elongation defects in SF3B1K700E are normalized by modulating epigenetic factors but 3 cryptic splicing events are not reversed - Targeting the Sin3/HDAC pathway to normalize RNAPII elongation defect is a potential therapeutic approach in these disorders GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/530019v3_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@dc6d22org.highwire.dtl.DTLVardef@180f36borg.highwire.dtl.DTLVardef@ed341forg.highwire.dtl.DTLVardef@f8dfb2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Epigenetic signature of human immune aging: the GESTALT study.

Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood borne immune cell types (naive B, naive CD4+and CD8+ T cells, granulocytes, monocytes and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousand of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In-silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1{beta}) and REST transcription factor motifs respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to transcription factor motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

molecular biology↗

bioRxiv2023

Development and qualification of a high-yield recombinant human Erythropoietin biosimilar

Recombinant human erythropoietin (rhEPO) has been saving millions of lives worldwide as a potent and safe treatment for the lack of erythrocyte, which is caused by chronic kidney disease (CKD) and other issues. Several biosimilars of rhEPO have been approved since the expiry of the relevant patents to provide cost-effective options but the price of rhEPO is still high for the affordability of global community. Therefore, development of biosimilar of rhEPO at a lower price is highly necessary. Here we report the development and characterization of a biosimilar of rhEPO with high-yield satisfying regulatory requirements. The hEPO-expressing cDNA was stably expressed in CHO cells with successive transfection. The master cell bank (MCB) and working cell bank (WCB) were established from the best selected clone and characterized for 50 passages. The rhEPO was expressed from the WCB in single-use suspension culture system with a high-titer (1.24{+/-}0.16 g/L). To the best of our knowledge this is the highest reported rhEPO titer to date. The rhEPO was purified using a series of validated chromatography unit processes including virus inactivation and filtration. The purified EPO was formulated in serum-free buffer, sterile filtered, and analyzed as the biosimilar of reference product Eprex(R). Physicochemical analysis strongly suggested similarities between the developed rhEPO (GBPD002) and the reference. The in vitro and in vivo functional assays confirmed the similar biofunctionality of the GBPD002 and Eprex(R). GBPD002 could provide a less-expensive solution to the needful communities as an effective and safe biosimilar where rhEPO treatment is necessary.

bioengineering↗

bioRxiv2023

A Cost-effective Purification Process for Erythropoietin Biosimilar through Downstream Process Engineering

Well-characterized and scalable downstream process for purification of biologics is extremely demanding for delivering quality therapeutics to patients at a reasonable price. Erythropoietin (EPO) is a blockbuster biologic with diverse clinical applications but its application is limited to financially well-off societies due to high price. The high price of EPO is associated with the technical difficulties related to the purification challenge to obtain qualified product with a cost-effective defined process. Though there are reports for purification of EPO but there is no report of well-characterized downstream process with critical process parameters (CPPs) that can deliver EPO consistently satisfying the quality target product profile (QTPP), which is a critical regulatory requirement. To advance the field, we applied quality by design (QbD) principle and design of experiment (DoE) protocol to establish an effective process, which is scalable up to 100x batch size satisfying QTPP. We have successfully transformed the process from static mode to dynamic mode and validated. Insignificant variation (p> 0.05) within and between 1x, 10x and 100x batches showed that the process is reproducible and seamlessly scalable. The biochemical analysis along with the biofunctionality data ensures that the products from different-scale batches were indifferent and comparable to a reference product. Our study thereby established a robust and scalable downstream process of EPO biosimilar satisfying QTPP. The technological scheme presented here can speed-up the production of not only EPO but many other life-saving biologics and make them available to mass population at a reduced cost.

bioengineering↗

bioRxiv2023

Weakly-supervised temporal segmentation of cell-cycle stages with center-cell focus using Recurrent Neural Networks

Training deep-learning models for biomedical images has always been a problem due to the lack of annotated data. Here we propose using a model and a training approach for the weakly-supervised temporal classification of cell-cycle stages during mitosis. Instead of using annotated data, by using an ordered set of classes called transcript, our proposed approach classifies the cell-cycle stages of cell video sequences. The network design helps to propagate information in time using Recurrent Neural Network and helps to focus the features on the center-cell. The algorithm is evaluated on four datasets from LiveCellMiner and has a performance close to the supervised approaches, which is impressive, considering that annotated data is not used in training.

bioinformatics↗

bioRxiv2022

Post-secretory synthesis of a natural analog of iron-gall ink in the black nectar of Melianthus spp.

The black nectar of Melianthus flowers is thought to serve as a visual attractant to pollinators, but the chemical identity and synthesis of the black pigment are unknown. Here we report that the black nectar contains a natural analog of iron-gall ink, which humans have used since medieval times. Specifically, dark black nectar at anthesis contains high levels of ellagic acid and iron; synthetic solutions of ellagic acid and iron(III) recapitulate the black color of the nectar. Conversely, lightly colored nectars before and after anthesis contain significantly lower levels of ellagic acid and iron, but higher levels of gallic acid. We then explored the possibility of post-secretory synthesis of ellagic acid from gallic acid. Indeed, Melianthus nectar contains a peroxidase that oxidizes gallic acid to form ellagic acid. Reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron can fully recreate the black color of the nectar. Visual modeling indicates that the black color is both visible and conspicuous to birds within the context of the flower. In summary, the black nectar of Melianthus is derived from an ellagic acid-Fe complex analogous to iron-gall ink and is likely involved in the attraction of passerine bird pollinators.

plant biology↗

bioRxiv2022

RNA conformational propensities determine cellular activity

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes 1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend not only on the strength of the contacts, but also on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities impact cellular activity. Here, we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

biophysics↗

bioRxiv2022

END-TO-END CLASSIFICATION OF CELL-CYCLE STAGES WITH CENTER-CELL FOCUS TRACKER USING RECURRENT NEURAL NETWORKS

Cell division, or mitosis, guarantees the accurate inheritance of the genomic information kept in the cell nucleus. Malfunctions in this process cause a threat to the health and life of the organism, including cancer and other manifold diseases. It is therefore crucial to study in detail the cell-cycle in general and mitosis in particular. Consequently, a large number of manual and semi-automated time-lapse microscopy image analyses of mitosis have been carried out in recent years. In this paper, we propose a method for automatic detection of cell-cycle stages using a recurrent neural network (RNN). An end-to-end model with center-cell focus tracker loss, and classification loss is trained. The evaluation was conducted on two time-series datasets, with 6-stages and 3-stages of cell splitting labeled. The frame-to-frame accuracy was calculated and precision, recall, and F1-Score were measured for each cell-cycle stage. We also visualized the learned feature space. Image reconstruction from the center-cell focus module was performed which shows that the network was able to focus on the center-cell and classify it simultaneously. Our experiments validate the superior performance of the proposed network compared to a classifier baseline.

bioinformatics↗

bioRxiv2022

Evolutionary dynamics of dengue virus in India

More than a hundred thousand dengue cases are diagnosed in India annually, and about half of the countrys population carries dengue virus-specific antibodies. Dengue propagates and adapts to the selection pressures imposed by a multitude of factors that can lead to the emergence of new variants. Yet, there has been no systematic analysis of the evolution of the dengue virus in the country. Here, we present a comprehensive analysis of all DENV gene sequences collected between 1956 and 2018 from India. We examine the spatio-temporal dynamics of India-specific genotypes, their evolutionary relationship with global and local dengue virus strains, interserotype dynamics and their divergence from the vaccine strains. Our analysis highlights the co-circulation of all DENV serotypes in India with cyclical outbreaks every 3-4 years. Since 2000, genotype III of DENV1, cosmopolitan genotype of DENV2, genotype III of DENV3 and genotype I of DENV4 have been dominating across the country. Substitution rates are comparable across the serotypes, suggesting a lack of serotype-specific evolutionary divergence. Yet, the envelope (E) protein displays strong signatures of evolution under immune selection. Apart from drifting away from its ancestors and other contemporary serotypes in general, we find evidence for recurring interserotype drift towards each other, suggesting selection via cross-reactive antibody-dependent enhancement. We identify the emergence of the highly divergent DENV4-Id lineage in South India, which has acquired half of all E gene mutations in the antigenic sites. Moreover, the DENV4-Id is drifting towards DENV1 and DENV3 clades, suggesting the role of cross-reactive antibodies in its evolution. Due to the regional restriction of the Indian genotypes and immunity-driven virus evolution in the country, [~]50% of all E gene differences with the current vaccines are focused on the antigenic sites. Our study shows how high incidence and pre-existing population immunity are shaping dengue virus evolution in India. Author summaryDengue is a mosquito-borne disease with four closely related serotypes of the virus (DENV1-4). Further, cross-reacting dengue antibodies from a previous infecting dengue serotype can protect or enhance infection from other serotypes. This can force the emergence of new dengue variants that find ways to escape the immune action or take advantage of it. In endemic countries like India, high rates of previous dengue infection can drive the evolution of dengue serotypes in complex ways. We compare all published dengue virus sequences to understand how new variants of dengue are emerging in India. Dengue cases and corresponding viruses display triennial surges. Further, the dengue envelope protein for each serotype shows recurring divergence and reversal towards its ancestral strain over a three-year window. Such fluctuations are also correlated among the dengue serotypes in India and could arise from the changing levels of cross-reactive antibodies. This, combined with the regional exchange of the virus among Asia-Pacific countries, has led to the emergence of India-specific DENV lineages, including a new DENV4 (Id) variant. This has also contributed to significant variations in the epitope regions of the current dengue viruses in India compared to the vaccines with implications for their efficacy.

evolutionary biology↗

bioRxiv2022

RGG-motif proteins regulate mRNA translation upon genotoxic stress

Genotoxic stress response (GSR) mediated by mRNA translation and decay regulation remains poorly explored. Here, we identify a unique role of yeast RGG-motif protein Scd6 and its human ortholog LSM14A in mRNA translation control upon hydroxyurea (HU)-mediated GSR. Scd6/LSM14A, but not all tested RGG-containing proteins, localize to HU-induced cytoplasmic puncta in an RGG-dependent manner. The absence of Scd6 increases HU tolerance but sensitizes the cells to HU upon overexpression of SRS2, a known dampener of the DNA- damage response. Scd6 binds SRS2 mRNA to repress its translation in cytoplasmic granules upon HU stress. Scd6-SRS2 interaction is modulated by arginine methylation (AM) and the LSm-domain, which acts as a cis-regulator of Scd6 AM. Polysome-profiling experiments indicate that LSM14A regulates the translation of NHEJ factor mRNAs such as LIG4 (DNL4 homolog) and RTEL1 (SRS2 functional homolog), and the NHEJ activity in response to HU. Overall, this report unveils the role of AM and Scd6/LSM14A in the GSR by determining the translation status of specific mRNAs.

cell biology↗

bioRxiv2022

Ion-channel degeneracy and heterogeneities in the emergence of complex spike bursts in CA3 pyramidal neurons

Complex spike bursting (CSB) is a characteristic electrophysiological signature exhibited by several neurons and has been implicated in neural plasticity, learning, perception, anesthesia, and active sensing. Here, we address the question of how pronounced intrinsic and synaptic heterogeneities affect CSB, with hippocampal CA3 pyramidal neurons (CA3PN) as a substrate where CSB emergence and heterogeneities are well-characterized. We randomly generated 12,000 unique models and found 236 valid models that satisfied 11 characteristic CA3PN measurements. These morphologically and biophysically realistic valid models accounted for gating kinetics and somato-dendritic expression profiles of 10 active ion channels. This heterogeneous population of valid models was endowed with broad distributions of underlying parameters showing weak pair-wise correlations. We found two functional subclasses of valid models, intrinsically bursting and regular spiking, with significant differences in the expression of calcium and calcium-activated potassium conductances. We triggered CSB in all 236 models through different intrinsic or synaptic protocols and observed considerable heterogeneity in CSB propensity and properties spanning models and protocols. Finally, we employed virtual knockout analyses and showed that synergistic interactions between intrinsic and synaptic mechanisms regulated CSB emergence and dynamics. Specifically, although there was a dominance of calcium and calcium-activated potassium channels in the emergence of CSB, individual deletion of none of the several ion channels or N-methyl-D-aspartate receptors resulted in the complete elimination of CSB across all models. Together, our analyses critically implicate ion-channel degeneracy in the robust emergence of CSB and other characteristic signatures of CA3PNs, despite pronounced heterogeneities in underlying intrinsic and synaptic properties.

neuroscience↗

bioRxiv2022

Nucleic acid quantification with amplicon yield in recombinase polymerase amplification

Amplification-based qPCR provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to different settings. Here, we adapted isothermal Recombinase Polymerase Amplification (RPA) reaction to develop a semi-quantitative method that relies on final amplicon yield to estimate initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and show that the amplicon yields at the end of the RPA reaction correlates well to the starting DNA concentration while reducing non-specific amplification robustly. We demonstrate this approach termed here as quantitative endpoint RPA (qeRPA) to detect DNA over five log orders with detection limit of 100 molecules. Using a linear regression model with normalized endpoint intensity (NEI) standard curve, we estimate viral load from the serum of dengue-infected patients with comparable performance to qPCR. Hence, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field-deployment in limited-resource settings.

biochemistry↗

bioRxiv2021

ESR2 regulates indian hedgehog signaling in neonatal rat ovary

The transcriptional regulatory function of estrogen receptor {beta} (ESR2) is essential for the regulation of primordial follicle activation (PFA). Increased PFA due to the loss of ESR2 becomes evident as early as postnatal day 8 (PND8). To identify the ESR2-regulated genes that control PFA, we performed RNA-seq analyses of wildtype, and Esr2 knockout (Esr2KO) neonatal rat ovaries collected on PND4, PND6, and PND8. Among the differentially expressed genes in Esr2KO ovaries, indian hedgehog (Ihh) displayed the highest downregulation among the ovary enriched genes. IHH regulated genes including Hhip as well as the steroidogenic enzymes were also downregulated in Esr2KO rat ovaries. Remarkably, the expression of Ihh in Esr2KO ovaries was not upregulated despite the high levels of Gdf9 and Bmp15, which are known regulators of Ihh expression in granulosa cells. Our findings suggest that indian hedgehog signaling in the neonatal rat ovary is dependent on ESR2.

developmental biology↗

bioRxiv2021

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha()-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the {beta}-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for {beta}-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the {beta}-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the {beta}-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the {beta}-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the {beta}-tongue and the tendency to induce disorder in the {beta}-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general. Author summaryBacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form bilayer spanning nanopores. Unregulated pore formation disrupts ionic balance that compromises the permeability of the cell, leading to cell death and infection. PFTs display remarkable structural plasticity that allow these proteins to interconvert between the two physiochemically distinct water soluble and membrane bound forms. However the molecular mechanism for this robust interconversion is poorly understood. For example, upon membrane binding, cytolysin A (ClyA), an -PFT, shows one of the largest conformational changes in protein structure among the PFT family of proteins. In order to understand this transtition we characterized several point mutations in ClyA using experiments and molecular dynamics (MD) simulations to understand the role of two essential ClyA motifs ({beta} tongue and N-terminus) implicated in the conformational changes responsible for oligomerization and conferring stability to the pore state. Our study reveals that the innate conformational flexibility of the {beta} tongue results in a disordered intermediate state that facilitate the complete transition to the pore state. This tendency to disorder is compromised to varying degrees in ClyA mutants, correlating with the loss of lytic activity. Our results suggest that the finely tuned conformational flexibility in the membrane motifs of ClyA are critical to its function, revealing a broader paradigm that could be at play during membrane associated secondary structure transitions of proteins in general.

biophysics↗