Biology subjects

Sharma, N.

Publications and source records attributed to Sharma, N..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Multi-environment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in allopolyploid Brassica napus

O_LICanola varieties exhibit discernible variation in drought avoidance and drought escape traits, suggesting its adaptation to water-deficit environments. However, the underlying mechanisms are poorly understood. C_LIO_LIA doubled haploid (DH) population was analysed to identify QTL associated with water use efficiency (WUE) related traits. Based on the resequenced parental genome data, we developed sequence-capture based markers for fine mapping. mRNA-Seq was performed to determine the expression of candidate genes underlying QTL for carbon isotope discrimination ({Delta}13C). C_LIO_LIQTL contributing to main and QTL x Environment interaction effects for {Delta}13C and for agronomic WUE were identified. One multi-trait QTL for {Delta}13C, days to flower, plant height and seed yield was identified on chromosome A09, in the vicinity of ERECTA. Interestingly, this QTL region was overlapped with a homoeologous exchange event (HE), suggesting its association with the major QTL. Transcriptome analysis revealed several differentially expressed genes between parental lines, including in HE regions. C_LIO_LIThis study provides insights into the complexity of WUE related genes in the context of canola adaptation to water-deficit conditions. Our results suggest that alleles for high {Delta}13C contribute positively to canola yield. Genetic and genomic resources developed herein could be utilised to make genetic gains for improving canola WUE. C_LI

plant biology↗

bioRxiv2021

Effect of prophylactic use of intra-nasal oil formulations in the hamster model of Covid-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in upper respiratory tract leading to coronavirus disease 2019 (Covid-19). Severe Covid-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting entry of the virus or its internalization in the upper respiratory tract, are of interest. Herein, we report the prophylactic application of two intra-nasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and Til tailya in SARS-CoV2 infection hamster model. Prophylactic nasal instillation of these oil formulations exhibited reduced viral load in lungs, and resulted in reduced body weight loss and pneumonitis. In line with reduced viral load, histopathlogical analysis revealed a reduction in lung pathology in Anu oil group as compared to the control infected group. However, Til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokines genes, including Th1 and Th17 cytokines for both the intra-nasal formulations as a result of decreased viral load. Together, the prophylactic intra-nasal application of Annu oil seems to be useful in limiting both the viral load and disease severity disease in SARS-CoV2 infection in hamster model.

microbiology↗

bioRxiv2021

Prediction of antibiotic resistant strains of bacteria from their beta-lactamases protein

Number of beta-lactamase variants have ability to deactivate ceftazidime antibiotic, which is the most commonly used antibiotic for treating infection by Gram-negative bacteria. In this study an attempt has been made to develop a method that can predict ceftazidime resistant strains of bacteria from amino acid sequence of beta-lactamases. We obtained beta-lactamases proteins from the {beta}-lactamase database, corresponding to 87 ceftazidime-sensitive and 112 ceftazidime-resistant bacterial strains. All models developed in this study were trained, tested, and evaluated on a dataset of 199 beta-lactamases proteins. We generate 9149 features for beta-lactamases using Pfeature and select relevant features using different algorithms in scikit-learn package. A wide range of machine learning techniques (like KNN, DT, RF, GNB, LR, SVC, XGB) has been used to develop prediction models. Our random forest-based model achieved maximum performance with AUROC of 0.80 on training dataset and 0.79 on the validation dataset. The study also revealed that ceftazidime-resistant beta-lactamases have amino acids with non-polar side chains in abundance. In contrast, ceftazidime-sensitive beta-lactamases have amino acids with polar side chains and charged entities in abundance. Finally, we developed a webserver "ABCRpred", for the scientific community working in the era of antibiotic resistance to predict the antibiotic resistance/susceptibility of beta-lactamase protein sequences. The server is freely available at (http://webs.iiitd.edu.in/raghava/abcrpred/). Key PointsO_LICeftazidime is commonly used to treat infection caused by Gram-negative bacteria. C_LIO_LIBeta-lactamase is responsible for lysing ceftazidime, make it resistant to bacteria. C_LIO_LIComparison of resistant and sensitive variants of beta-lactamase. C_LIO_LIClassification of sensitive and resistant strain of bacteria based on beta-lactamase. C_LIO_LIPrediction models have been developed using different machine learning techniques. C_LI

bioinformatics↗

bioRxiv2021

Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin

Dystonia is a neurologic disorder associated with an increasingly large number of variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is a critical step in understanding the key pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations in isogenic induced pluripotent stem cells (iPSCs). We used this strategy to investigate the molecular consequences of variation in THAP1, which encodes a transcription factor that has been linked to neural differentiation. Multiple pathogenic mutations that have been associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter its DNA binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight mutations in a common iPSC background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling of these neural derivatives followed by joint analysis of the most robust individual signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. Based on these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to tissue from control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.

genetics↗

bioRxiv2021

ChAlPred: A Web Server for Prediction of Allergenicity of Chemical Compounds

Allergy is the abrupt reaction of the immune system that may occur after the exposure with allergens like protein/peptide or chemical allergens. In past number of methods of have been developed for classifying the protein/peptide based allergen. To the best of our knowledge, there is no method to classify the allergenicity of chemical compound. Here, we have proposed a method named "ChAlPred", which can be used to fill the gap for predicting the chemical compound that might cause allergy. In this study, we have obtained the dataset of 403 allergen and 1074 non-allergen chemical compounds and used 2D, 3D and FP descriptors to train, test and validate our prediction models. The fingerprint analysis of the dataset indicates that PubChemFP129 and GraphFP1014 are more frequent in the allergenic chemical compounds, whereas KRFP890 is highly present in non-allergenic chemical compounds. Our XGB based model achieved the AUC of 0.89 on validation dataset using 2D descriptors. RF based model has outperformed other classifiers using 3D descriptors (AUC = 0.85), FP descriptors (AUC = 0.92), combined descriptors (AUC = 0.93), and hybrid model (AUC = 0.92) on validation dataset. In addition, we have also reported some FDA-approved drugs like Cefuroxime, Spironolactone, and Tioconazole which can cause the allergic symptoms. A user user-friendly web server named "ChAlPred" has been developed to predict the chemical allergens. It can be easily accessed at https://webs.iiitd.edu.in/raghava/chalpred/.

immunology↗

bioRxiv2021

Development of a rapid LC-MS/MS method for the simultaneous quantification of various flavonoids and phytohormones extracted from Medicago Truncatula leaves

Flavonoids are small metabolites of plants, which are involved in the regulation of plant development as well as defence against pathogens. Quantitation of these flavonoids in plant samples is highly important and essential in the food and herbal industry. Hence, robust, reliable and sensitive methods are required for the analysis of these compounds in plant samples. In the present study, a high performance liquid chromatographic-tandem mass spectrometric (HPLC- ESI-MS/MS) method was developed and validated for the determination of nine flavonoids, including Liquiritigenin, Naringenin, Genistein, Daidzein, Formononetin, Biochanin A, 2-hydroxy formononetin, 2-methoxyformononetin, Medicarpin, and two phytohormones, Salicylic acid and Jasmonic acid, in Medicago truncatula. The analytes were separated by means of C-18, reversed-phase chromatography and detected using QTRAP mass spectrometer. Molecules were quantified using different transitions in positive and negative ion modes simultaneously in 12 minutes. The on-column limit of detection of all the analytes was as low as 0.03 pg, whereas the limit of quantification of all the compounds was observed upto 0.1 pg levels. Further the method was also validated in terms of specificity, linearity (r2> 0.99), average recovery (90.6 - 110.3%), accuracy (RE% [≤] 3%) and precision (RSD% [≤] 3%). As a proof of concept, the developed method was successfully used for the quantitation of these flavonoids and phytohormones from leaf extract of Medicago truncatula.

biochemistry↗

bioRxiv2021

Mechanism of transcription modulation by the transcription-repair coupling factor

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes distressed RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

biophysics↗

bioRxiv2021

Effect of landscape heterogeneity on diurnal raptor community richness and diversity in Jammu Shiwaliks, Jammu and Kashmir

In this study, we examined the richness and diversity of diurnal raptors along with their foraging guilds across different land uses in a sub-tropical landscape during December 2016 to November 2018. A total of 80 vantage points, 19 line transects and 36 road transects were sampled in 33 sites in six different habitat types in the study area where we recorded 3409 individuals of 29 diurnal raptors in 2 orders and 3 families. Significant variation in bird abundance was observed among different habitat types, farmlands being more specious followed by pure forests, water bodies and forest-farmland interfaces. Among the seasons, summers recorded higher abundance followed by winter, monsoon and post-monsoon. A low diversity value (H'=2.22) however was observed for the whole study area with mean monthly highest recorded during February (H'=2.44) and least during June (H'=1.85). Most of the raptors observed for their food types and foraging were predators (n=22) and rest were carrion feeders (n=22). Fourteen among all observed diurnal raptors were winter visitors and 13 residents with 9 reported globally threatened. A moderately high richness of diurnal raptors substantiate high conservation value of these habitats especially the forest patches and farmlands and thus calls for effective management strategies for the conservation and proliferation of raptors in sub-tropical areas of Jammu region.

ecology↗

bioRxiv2021

Osa-miR820 regulatory node primes rice plants to tolerate salt stress in an agronomically advantageous manner

Plant microRNAs (miRs) play an important role in regulating gene expression under normal and stressful environments. Here we report the functional implications on the role of Osa-miR820, which can be grouped as a young, rice-specific miR. It is a member of the class II transposon-derived small RNA family and is processed as 21-nt and 24-nt length variants, respectively. Size of the miR820 family varies from 1 to 16 across the Oryza AA genomes. The 21-nt Osa-miR820 negatively regulates a de novo methylase, OsDRM2 (domains rearranged methyl transferase) that prevents methylation of the CACTA transposon loci in the rice genome. In an earlier report we have detailed the expression profiles of Osa-miR820 and its target in abiotic stress responses using rice varieties exhibiting varying response to salt stress. In this study, artificial miR based approach was employed to specifically overexpress 21-nt Osa-miR820 in rice plants (OX-820). These plants exhibited enhanced vigour, ~25-30% increase in the number of spikelets per panicle and increased grain filling, under normal and salt stress conditions. The OX-820 lines showed a better water use efficiency and higher proline accumulation under salt stress. These plants can serve as a useful source for dissecting the molecular machinery governed by Osa-miR820:DRM2 node to prime tolerance to salt stress in an agronomically advantageous manner.

plant biology↗

bioRxiv2020

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substrates in vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear. In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

biochemistry↗

bioRxiv2020

KNOX HOMOLOGS SHOOT MERISTEMLESS (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during embryonic meristem development in Arabidopsis thaliana

In Arabidopsis, the genes SHOOT MERISTEMLESS (STM) and CLAVATA3 (CLV3) antagonistically regulate shoot meristem development. STM is essential for both development and maintenance of the meristem, as stm mutants fail to develop a shoot meristem during embryogenesis. CLV3, on the other hand, negatively regulates meristem proliferation, and clv3 mutants possess an enlarged shoot meristem. Genetic interaction studies revealed that stm and clv3 dominantly suppress each others phenotypes. STM works in conjunction with its closely related homologue KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6) to promote meristem development and organ separation, as stm knat6 double mutants fail to form a meristem and produce a fused cotyledon. In this study, we show that clv3 fails to promote post-embryonic meristem formation in stm-1 background if we also remove KNAT6. stm-1 knat6 clv3 triple mutants result in early meristem termination and produce fused cotyledons similar to stm knat6 double mutant. Notably, the stm-1 knat6 and stm-1 knat6 clv3 alleles lack tissue in the presumed region of SAM. stm knat6 clv3 also showed reduced inflorescence size and shoot apex size as compared to clv3 single or stm clv3 double mutants. In contrast to previously published data, these data suggest that stm is epistatic to clv3 in postembryonic meristem development. HighlightSTM and KNAT6 genes determine post-embryonic meristem formation and activity in Arabidopsis. clv3 mutation is unable to rescue the stm knat6 meristemless phenotype.

plant biology↗

bioRxiv2020

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.

systems biology↗

bioRxiv2020

Tuning of self-renewing capacity in theArabidopsis stomatal lineage by hormonaland nutrition regulation of asymmetric celldivisions

Asymmetric and self-renewing divisions build and pattern tissues. In the Arabidopsis thaliana stomatal lineage, asymmetric cell divisions, guided by polarly localized cortical proteins, generate the majority of cells on the leaf surface. These divisions can be fine-tuned by systemic and environmental signals to modify tissue development, but the molecular mechanisms by which plants incorporate such cues to regulate asymmetric divisions are largely unknown. In a screen for modulators of cell polarity and asymmetric divisions, we identified a mutation in CONSTITIUTIVE TRIPLE RESPONSE 1, a negative regulator of ethylene signaling. We subsequently revealed antagonistic impacts of ethylene and glucose signaling on the self-renewing capacity of stomatal lineage stem cells. Quantitative analysis of the impacts of these signaling systems on cell polarity and fate dynamics showed that developmental information may be encoded in both the spatial and temporal asymmetries of polarity proteins. Taken together, our results provide a framework for a mechanistic understanding of how systemic information such as nutritional status and environmental factors tune stem cell behavior in the stomatal lineage, ultimately enabling optimization of leaf size and cell-type composition.

plant biology↗

bioRxiv2020

Single-Cell Resolution of Lineage Trajectories in the Arabidopsis Stomatal Lineage and Developing Leaf

Dynamic cell states underlie flexible developmental programs, such as with the stomatal lineage of the Arabidopsis epidermis. Initial stages of the lineage feature asynchronous and indeterminate divisions modulated by environmental cues, enabling cell fate flexibility to generate the requisite density and pattern of stomata for a given environment. It remains unclear, however, how flexibility of cell fates is controlled. Here, we uncovered distinct models of cell state differentiation within Arabidopsis leaf tissue by leveraging single-cell transcriptomics and molecular genetics. Our findings resolved underlying heterogeneity within cell states of the flexible epidermal stomatal lineage, which appear to exist along a continuum, with progressive cell specification. Beyond the early stages of the lineage, we discovered that the core transcriptional regulator SPEECHLESS is required for cell fate commitment to yield stomatal guard cells. Overall, our work has refined the stomatal lineage paradigm and uncovered progressive cell state decisions along lineage trajectories in developing leaves.

plant biology↗

bioRxiv2020

Transient delivery of A-C/EBP protein perturbs differentiation of 3T3-L1 cells and induces preadipocyte marker genes

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves timely appearance of numerous transcription factors (TFs), foremost among them C/EBP{beta}, is expressed during early phases of differentiation. Here we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBP{beta} by A-C/EBP protein inhibitor. Signals from tagged A-C/EBP were observed in 3T3-L1 cells within 2hrs of protein inhibitor transfections whereas for gene transfection signals appeared in 48hrs. Following transient transfections, expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocytes pathways was analyzed. mRNA and protein expression profiles of adipocyte-marker genes showed lower expression in both A-C/EBP protein and gene transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between protein and gene transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfr, Pdgfr{beta}, Ly6A, CD34 Itgb1 showed high expression in protein samples whereas only Ly6A and CD34 were expressed in gene transfected samples. Pdgfr and Pdgfr{beta}, two known cell fate markers were expressed in protein transfected samples 5-days post-differentiation suggesting a possible reversal of differentiation. Our study provides evidences for the robust and efficient knockdown of C/EBP{beta} protein to understand time-dependent gene regulation during adipogenesis.

molecular biology↗

bioRxiv2020

Docking studies in targeting proteins involved in cardiovascular disorders using phytocompounds from Terminalia arjuna

Terminalia arjuna (Roxb.) Wight and Arnot (T. arjuna) commonly known as Arjuna has been known for its cardiotonic nature in heart failure, ischemic, cardiomyopathy, atherosclerosis, myocardium necrosis and also has been used in the treatment of different human disorders such as blood diseases, anaemia and viral diseases. Our focus has been on phytochemicals which do not exhibit any cytotoxicity and have significant cardioprotective activity. Since Protein-Ligand interactions play a key role in structure-based drug design, therefore with the help of molecular docking, we screened 19 phytochemicals present in T. arjuna and investigated their binding affinity against different cardiovascular target proteins. The three-dimensional (3D) structure of target cardiovascular proteins were retrieved from Protein Data Bank, and docked with 3D Pubchem structures of 19 phytochemicals using Autodock vina. Molecular docking and drug-likeness studies were made using ADMET properties while Lipinskis rule of five was performed for the phytochemicals to evaluate their cardio protective activity. Among all selected phytocompounds, arjunic acid, arjungenin, and terminic acid were found to fulfill all ADMET rules, drug likeness, and are less toxic in nature. Our studies, therefore revealed that these three phytochemicals from T. arjuna can be used as promising candidates for developing broad spectrum drugs against cardiovascular diseases.

bioinformatics↗

bioRxiv2020

The First complete Zoroastrian-Parsi Mitochondria Reference Genome: Implications of mitochondrial signatures in an endogamous, non-smoking population

The present-day Zoroastrian-Parsis have roots in ancient pastoralist migrations from circumpolar regions leading to their settlement on the Eurasian Steppes and later, as Indo-Iranians in the Fertile Crescent. After migrating from the Persian province of Pars to India, the Zoroastrians from Pars ("Parsis") practiced endogamy, thereby preserving their genetic identity and social practices. The study was undertaken to gain an insight into the genetic consequences of migration on the community, the practice of endogamy, to decipher the phylogenetic relationships with other groups, and elucidate the disease linkages to their individual haplotypes We generated the de novo the Zoroastrian-Parsi Mitochondrial Reference Genome (AGENOME-ZPMS-HV2a-1), which is the first complete mitochondrial reference genome assembled for this group. Phylogenetic analysis of an additional 99 Parsi mitochondrial genome sequences showed the presence of HV, U, T, A and F (belonging to the macrohaplogroup N) and Z and other M descendents of the macrohaplogroup M (M5, M39, M33, M4452, M24, M3, M30, M2, M430, M2, M35 and M27) and a largely Persian origin for the Parsi community. We assembled individual reference genomes for each major haplogroup and the Zoroastrian-Parsi Mitochondrial Consensus Genome (AGENOME-ZPMCG V1.0), which is the first consensus genome assembled for this group. We report the existence of 420 mitochondrial genetic variants, including 12 unique variants, in the 100 Zoroastrian-Parsi mitochondrial genome sequences. Disease association mapping showed 217 unique variants linked to longevity and 41 longevity-associated disease phenotypes across the majority of haplogroups. Analysis of the coding genes, tRNA genes, and the D-loop region revealed haplogroup-specific disease associations for Parkinsons disease, Alzheimers disease, cancers, and rare diseases. No known mutations linked to lung cancer were found in our study. Mutational signatures linked to tobacco carcinogens, specifically, the C>A and G>T transitions, were observed at extremely low frequencies in the Parsi cohort, suggestive of an association between the cultural norm prohibiting smoking and its reflection in the genetic signatures. In sum, the Parsi mitochondrial genome provides an exceptional resource for determining details of their migration and uncovering novel genetic signatures for wellness and disease.

genomics↗

bioRxiv2020

Comparative MD simulations and advanced analytics based studies on wild-type and hot-spot mutant A59G HRas

The Ras family of proteins is known to play an important role in cellular signal transduction. The oncoprotein Ras is also found to be mutated in [~]90% of the pancreatic cancers, of which G12V, G13V, A59G and Q61L are the known hot-spot mutants. These ubiquitous proteins fall in the family of G-proteins, and hence switches between active GTP bound and inactive GDP bound states, which is hindered in most of its oncogenic mutant counterparts. Moreover, Ras being a GTPase has an intrinsic property to hydrolyze GTP to GDP, which is obstructed due to mutations and lends the mutants stuck in constitutively active state leading to oncogenic behavior. In this regard, the present study aims to understand the dynamics involved in the hot-spot mutant A59G-Ras using long classical MD simulations ([~]10{micro}s) and comparing the same with its wild-type counterpart. Advanced analytics using Markov State Model (MSM) based approach has been deployed to comparatively understand the transition path for the wild-type and mutant systems. Roles of crucial residues like Tyr32, Gln61 and Tyr64 have also been established using multivariate PCA analyses. Furthermore, this multivariate PCA analysis also provides crucial features which may be used as reaction coordinates for biased simulations for further studies. The absence of formation of pre-hydrolysis network is also reported for the mutant conformation, using the distance-based analyses (between crucial residues) of the conserved regions. The implications of this study strengthen the hypothesis that the disruption of the pre-hydrolysis network in the mutant A59G ensemble might lead to permanently active oncogenic conformation in the mutant conformers.

biophysics↗