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Naganathan Athi N.
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Naganathan Athi N.
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Naganathan Athi N.
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Naganathan, Athi N.
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17 results
Now showing 1 - 10 of 17
- PublicationProtein plasticity driven by disorder and collapse governs the heterogeneous binding of CytR to DNA(04-05-2018)
;Munshi, Sneha ;Gopi, Soundhararajan ;Subramanian, Sandhyaa ;Campos, Luis A.The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-to-globule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the single-molecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation. - PublicationErratum to “Thermodynamics and folding landscapes of large proteins from a statistical mechanical model†[Current Research in Structural Biology 1 (2019) 6–12] (Current Research in Structural Biology (2019) 1 (6–12), (S2665928X19300030), (10.1016/j.crstbi.2019.10.002))(01-01-2020)
;Gopi, Soundhararajan ;Aranganathan, AkashnathanThe Publisher regrets that the “Conflict of Interest” statement was not included in the published article at the time of publication. The Authors confirm that they do not have any Conflict of interest to report for this article. The Publisher would like to apologise for any inconvenience caused. - PublicationToward a quantitative description of microscopic pathway heterogeneity in protein folding(01-01-2017)
;Gopi, Soundhararajan ;Singh, Animesh ;Suresh, Swaathiratna ;Paul, Suvadip ;Ranu, SayanHow many structurally different microscopic routes are accessible to a protein molecule while folding? This has been a challenging question to address experimentally as single-molecule studies are constrained by the limited number of observed folding events while ensemble measurements, by definition, report only an average and not the distribution of the quantity under study. Atomistic simulations, on the other hand, are restricted by sampling and the inability to reproduce thermodynamic observables directly. We overcome these bottlenecks in the current work and provide a quantitative description of folding pathway heterogeneity by developing a comprehensive, scalable and yet experimentally consistent approach combining concepts from statistical mechanics, physical kinetics and graph theory. We quantify the folding pathway heterogeneity of five single-domain proteins under two thermodynamic conditions from an analysis of 100.000 folding events generated from a statistical mechanical model incorporating the detailed energetics from more than a million conformational states. The resulting microstate energetics predicts the results of protein engineering experiments, the thermodynamic stabilities of secondary-structure segments from NMR studies, and the end-to-end distance estimates from single-molecule force spectroscopy measurements. We find that a minimum of ∼3-200 microscopic routes, with a diverse ensemble of transition-path structures, are required to account for the total folding flux across the five proteins and the thermodynamic conditions. The partitioning of flux amongst the numerous pathways is shown to be subtly dependent on the experimental conditions that modulate protein stability, topological complexity and the structural resolution at which the folding events are observed. Our predictive methodology thus reveals the presence of rich ensembles of folding mechanisms that are generally invisible in experiments, reconciles the contradictory observations from experiments and simulations and provides an experimentally consistent avenue to quantify folding heterogeneity. - PublicationStructural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble(23-02-2022)
;Golla, Hemashree ;Kannan, Adithi ;Gopi, Soundhararajan ;Murugan, Sowmiya ;Perumalsamy, Lakshmi R.The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural-energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical-mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge-charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a “gatekeeper”, modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge-charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains. - PublicationPPerturb: A Server for Predicting Long-Distance Energetic Couplings and Mutation-Induced Stability Changes in Proteins via Perturbations(21-01-2020)
;Gopi, Soundhararajan ;Devanshu, Devanshu ;Rajasekaran, Nandakumar ;Anantakrishnan, SathvikThe strength of intraprotein interactions or contact network is one of the dominant factors determining the thermodynamic stabilities of proteins. The nature and the extent of connectivity of this network also play a role in allosteric signal propagation characteristics upon ligand binding to a protein domain. Here, we develop a server for rapid quantification of the strength of an interaction network by employing an experimentally consistent perturbation approach previously validated against a large data set of 375 mutations in 19 different proteins. The web server can be employed to predict the extent of destabilization of proteins arising from mutations in the protein interior in experimentally relevant units. Moreover, coupling distances - a measure of the extent of percolation on perturbation - and overall perturbation magnitudes are predicted in a residue-specific manner, enabling a first look at the distribution of energetic couplings in a protein or its changes upon ligand binding. We show specific examples of how the server can be employed to probe for the distribution of local stabilities in a protein, to examine changes in side chain orientations or packing before and after ligand binding, and to predict changes in stabilities of proteins upon mutations of buried residues. The web server is freely available at http://pbl.biotech.iitm.ac.in/pPerturb and supports recent versions of all major browsers. - PublicationExtracting the Hidden Distributions Underlying the Mean Transition State Structures in Protein Folding(05-04-2018)
;Gopi, Soundhararajan ;Paul, Suvadip ;Ranu, SayanThe inherent conflict between noncovalent interactions and the large conformational entropy of the polypeptide chain forces folding reactions and their mechanisms to deviate significantly from chemical reactions. Accordingly, measures of structure in the transition state ensemble (TSE) are strongly influenced by the underlying distributions of microscopic folding pathways that are challenging to discern experimentally. Here, we present a detailed analysis of 150,000 folding transition paths of five proteins at three different thermodynamic conditions from an experimentally consistent statistical mechanical model. We find that the underlying TSE structural distributions are rarely unimodal, and the average experimental measures arise from complex underlying distributions. Unfolding pathways also exhibit subtle differences from folding counterparts due to a combination of Hammond behavior and native-state movements. Local interactions and topological complexity, to a lesser extent, are found to determine pathway heterogeneity, underscoring the importance of the balance between local and nonlocal energetics in protein folding. - PublicationPStab: Prediction of stable mutants, unfolding curves, stability maps and protein electrostatic frustration(01-03-2018)
;Gopi, Soundhararajan ;Devanshu, Devanshu ;Krishna, PraveenWe present a web-server for rapid prediction of changes in protein stabilities over a range of temperatures and experimental conditions upon single- or multiple-point substitutions of charged residues. Potential mutants are identified by a charge-shuffling procedure while the stability changes (i.e. an unfolding curve) are predicted employing an ensemble-based statistical-mechanical model. We expect this server to be a simple yet detailed tool for engineering stabilities, identifying electrostatically frustrated residues, generating local stability maps and in constructing fitness landscapes. - PublicationA binding cooperativity switch driven by synergistic structural swelling of an osmo-regulatory protein pair(01-12-2019)
;Narayan, Abhishek ;Gopi, Soundhararajan ;Fushman, DavidUropathogenic E. coli experience a wide range of osmolarity conditions before and after successful infection. Stress-responsive regulatory proteins in bacteria, particularly proteins of the Hha family and H-NS, a transcription repressor, sense such osmolarity changes and regulate transcription through unknown mechanisms. Here we use an array of experimental probes complemented by molecular simulations to show that Cnu, a member of the Hha protein family, acts as an exquisite molecular sensor of solvent ionic strength. The osmosensory behavior of Cnu involves a fine-tuned modulation of disorder in the fourth helix and the three-dimensional structure in a graded manner. Order-disorder transitions in H-NS act synergistically with molecular swelling of Cnu contributing to a salt-driven switch in binding cooperativity. Thus, sensitivity to ambient conditions can be imprinted at the molecular level by tuning not just the degree of order in the protein conformational ensemble but also through population redistributions of higher-order molecular complexes. - PublicationThermodynamics and folding landscapes of large proteins from a statistical mechanical model(01-11-2019)
;Gopi, Soundhararajan ;Aranganathan, AkashnathanStatistical mechanical models that afford an intermediate resolution between macroscopic chemical models and all-atom simulations have been successful in capturing folding behaviors of many small single-domain proteins. However, the applicability of one such successful approach, the Wako-Saitô-Muñoz-Eaton (WSME) model, is limited by the size of the protein as the number of conformations grows exponentially with protein length. In this work, we surmount this size limitation by introducing a novel approximation that treats stretches of 3 or 4 residues as blocks, thus reducing the phase space by nearly three orders of magnitude. The performance of the ‘bWSME’ model is validated by comparing the predictions for a globular enzyme (RNase H) and a repeat protein (IκBα), against experimental observables and the model without block approximation. Finally, as a proof of concept, we predict the free-energy surface of the 370-residue, multi-domain maltose binding protein and identify an intermediate in good agreement with single-molecule force-spectroscopy measurements. The bWSME model can thus be employed as a quantitative predictive tool to explore the conformational landscapes of large proteins, extract the structural features of putative intermediates, identify parallel folding paths, and thus aid in the interpretation of both ensemble and single-molecule experiments. - PublicationA Disordered Loop Mediates Heterogeneous Unfolding of an Ordered Protein by Altering the Native Ensemble(20-08-2020)
;Bhattacharjee, Kabita ;Gopi, SoundhararajanThe high flexibility of long disordered or partially structured loops in folded proteins allows for entropic stabilization of native ensembles. Destabilization of such loops could alter the native ensemble or promote alternate conformations within the native ensemble if the ordered regions themselves are held together weakly. This is particularly true of downhill folding systems that exhibit weak unfolding cooperativity. Here, we combine experimental and computational methods to probe the response of the native ensemble of a helical, downhill folding domain PDD, which harbors an 11-residue partially structured loop, to perturbations. Statistical mechanical modeling points to continuous structural changes on both temperature and mutational perturbations driven by entropic stabilization of partially structured conformations within the native ensemble. Long time-scale simulations of the wild-type protein and two mutants showcase a remarkable conformational switching behavior wherein the parallel helices in the wild-type protein sample an antiparallel orientation in the mutants, with the C-terminal helix and the loop connecting the helices displaying high flexibility, disorder, and non-native interactions. We validate these computational predictions via the anomalous fluorescence of a native tyrosine located at the interface of the helices. Our observations highlight the role of long loops in determining the unfolding mechanisms, sensitivity of the native ensembles to mutational perturbations and provide experimentally testable predictions that can be explored in even two-state folding systems.