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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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9 results
Now showing 1 - 9 of 9
- PublicationGraded structural polymorphism in a bacterial thermosensor Protein(18-01-2017)
;Narayan, Abhishek ;Campos, Luis A. ;Bhatia, Sandhya ;Fushman, DavidThermosensing is critical for the expression of the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family. - PublicationSwitching Protein Conformational Substates by Protonation and Mutation(13-12-2018)
;Narayan, AbhishekProtein modules that regulate the availability and conformational status of transcription factors determine the rapidity, duration, and magnitude of cellular response to changing conditions. One such system is the single-gene product Cnu, a four-helix bundle transcription co-repressor, which acts as a molecular thermosensor regulating the expression of virulence genes in enterobacteriaceae through modulation of its native conformational ensemble. Cnu and related genes have also been implicated in pH-dependent expression of virulence genes. We hypothesize that protonation of a conserved buried histidine (H45) in Cnu promotes large electrostatic frustration, thus disturbing the H-NS, a transcription factor, binding face. Spectroscopic and calorimetric methods reveal that H45 exhibits a suppressed pKa of ∼5.1, the protonation of which switches the conformation to an alternate native ensemble in which the fourth helix is disordered. The population redistribution can also be achieved through a mutation H45V, which does not display any switching behavior at pH values greater than 4. The Wako-Saitô-Muñoz-Eaton (WSME) statistical mechanical model predicts specific differences in the conformations and fluctuations of the fourth and first helices of Cnu determining the observed pH response. We validate these predictions through fluorescence lifetime measurements of a sole tryptophan, highlighting the presence of both native and non-native interactions in the regions adjoining the binding face of Cnu. Our combined experimental-computational study thus shows that Cnu acts both as a thermo- and pH-sensor orchestrated via a subtle but quantifiable balance between the weak packing of a structural element and protonation of a buried histidine that promotes electrostatic frustration. - PublicationEvidence for the sequential folding mechanism in RNase H from an ensemble-based model(15-05-2014)
;Narayan, AbhishekThe number of distinct protein folding pathways starting from an unfolded ensemble, and hence, the folding mechanism is an intricate function of protein size, sequence complexity, and stability conditions. This has traditionally been a contentious issue particularly because of the ensemble nature of conventional experiments that can mask the complexity of the underlying folding landscape. Recent hydrogen-exchange experiments combined with mass spectrometry (HX-MS) reveal that the folding of RNase H proceeds in a hierarchical fashion with distinct intermediates and following a single discrete path. In our current work, we provide computational evidence for this unique folding mechanism by employing a structure-based statistical mechanical model. Upon calibrating the energetic terms of the model with equilibrium measurements, we predict multiple intermediate states in the folding of RNase H that closely resemble experimental observations. Remarkably, a simplified landscape representation adequately captures the folding complexity and predicts the possibility of a well-defined sequence of folding events. We supplement the statistical model study with both explicit solvent molecular simulations of the helical units and electrostatic calculations to provide structural and energetic insights into the early and late stages of RNase H folding that hint at the frustrated nature of its folding landscape. © 2014 American Chemical Society. - 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. - PublicationThermally versus Chemically Denatured Protein States(28-05-2019)
;Narayan, Abhishek ;Bhattacharjee, KabitaProtein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, UT and UD for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone-backbone interactions and identity versus backbone-solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients. - PublicationQuantifying Protein Disorder through Measures of Excess Conformational Entropy(19-05-2016)
;Rajasekaran, Nandakumar ;Gopi, Soundhararajan ;Narayan, AbhishekIntrinsically disordered proteins (IDPs) and proteins with a large degree of disorder are abundant in the proteomes of eukaryotes and viruses, and play a vital role in cellular homeostasis and disease. One fundamental question that has been raised on IDPs is the process by which they offset the entropic penalty involved in transitioning from a heterogeneous ensemble of conformations to a much smaller collection of binding-competent states. However, this has been a difficult problem to address, as the effective entropic cost of fixing residues in a folded-like conformation from disordered amino acid neighborhoods is itself not known. Moreover, there are several examples where the sequence complexity of disordered regions is as high as well-folded regions. Disorder in such cases therefore arises from excess conformational entropy determined entirely by correlated sequence effects, an entropic code that is yet to be identified. Here, we explore these issues by exploiting the order-disorder transitions of a helix in Pbx-Homeodomain together with a dual entropy statistical mechanical model to estimate the magnitude and sign of the excess conformational entropy of residues in disordered regions. We find that a mere 2.1-fold increase in the number of allowed conformations per residue (∼0.7kBT favoring the unfolded state) relative to a well-folded sequence, or ∼2N additional conformations for a N-residue sequence, is sufficient to promote disorder under physiological conditions. We show that this estimate is quite robust and helps in rationalizing the thermodynamic signatures of disordered regions in important regulatory proteins, modeling the conformational folding-binding landscapes of IDPs, quantifying the stability effects characteristic of disordered protein loops and their subtle roles in determining the partitioning of folding flux in ordered domains. In effect, the dual entropy model we propose provides a statistical thermodynamic basis for the relative conformational propensities of amino acids in folded and disordered environments in proteins. Our work thus lays the foundation for understanding and quantifying protein disorder through measures of excess conformational entropy. - PublicationTuning the Continuum of Structural States in the Native Ensemble of a Regulatory Protein(06-04-2017)
;Narayan, AbhishekThe mesoscale nature of proteins allows for an efficient coupling between environmental cues and conformational changes, enabling their function as molecular transducers. Delineating the precise structural origins of such a connection and the expected spectroscopic response has, however, been challenging. In this work, we perform a combination of urea-temperature double perturbation experiments and theoretical modeling to probe the conformational landscape of Cnu, a natural thermosensor protein. We observe unique ensemble signatures that point to a continuum of conformational substates in the native ensemble and that respond intricately to perturbations upon monitoring secondary and tertiary structures, distances between an intrinsic FRET pair, and hydrodynamic volumes. Binding assays further reveal a weakening of the Cnu functional complex with temperature, highlighting the molecular origins of signal transduction critical for pathogenic response in enterobacteriaceae. - PublicationFolding Intermediates, Heterogeneous Native Ensembles and Protein Function(03-12-2021)
; ;Dani, Rahul ;Gopi, Soundhararajan ;Aranganathan, AkashnathanNarayan, AbhishekSingle domain proteins fold via diverse mechanisms emphasizing the intricate relationship between energetics and structure, which is a direct consequence of functional constraints and demands imposed at the level of sequence. On the other hand, elucidating the interplay between folding mechanisms and function is challenging in large proteins, given the inherent shortcomings in identifying metastable states experimentally and the sampling limitations associated with computational methods. Here, we show that free energy profiles and surfaces of large systems (>150 residues), as predicted by a statistical mechanical model, display a wide array of folding mechanisms with ubiquitous folding intermediates and heterogeneous native ensembles. Importantly, residues around the ligand binding or enzyme active site display a larger tendency to partially unfold and this manifests as intermediates or excited states along the folding coordinate in ligand binding domains, transcription repressors, and representative enzymes from all the six classes, including the SARS-CoV-2 receptor binding domain (RBD) of the spike protein and the protease Mpro. It thus appears that it is relatively easier to distill the imprints of function on the folding landscape of larger proteins as opposed to smaller systems. We discuss how an understanding of energetic-entropic features in ordered proteins can pinpoint specific avenues through which folding mechanisms, populations of partially structured states and function can be engineered. - PublicationElectrostatic Frustration Shapes Folding Mechanistic Differences in Paralogous Bacterial Stress Response Proteins(07-08-2020)
;Narayan, Abhishek ;Gopi, Soundhararajan ;Lukose, BincyParalogous proteins play a vital role in evolutionary adaptation of organisms and species divergence. One outstanding question is the molecular basis for how folding mechanisms differ in paralogs that not only exhibit similar topologies but also evolve under near-identical selection pressures. Here, we address this question by studying a paralogous protein pair from enterobacteria, Hha and Cnu, combining experiments, simulations and statistical modeling. We find that Hha is less stable and folds an order of magnitude slower than Cnu despite similar packing and topological features. Differences in surface charge–charge interactions, however, promote a N-terminal biased unfolding mechanism in Hha unlike Cnu that unfolds via the C terminus. Our work highlights how electrostatic frustration contributes to the population of heterogeneous native ensembles in paralogs and the avenues through which evolutionary topological constraints could be overcome by modulating charge–charge interactions.