Anindita Das

All Publications

• Mechanism of Gating and Isoform-Specific Inhibition in Renal CLC Chloride Channels. bioRxiv : the preprint server for biology Chien, C. T., Sobecks, B. L., Powers, A. S., Kreiter, J., Das, A., Barry, C. N., Chen, M., Hinman, A., Petrakian, C. F., Trifkovic, N., Williams, B., Wood, C. A., Xu, M., Dror, R. O., Chiu, W., Maduke, M. 2026

  Abstract

  Hyponatremia is a prevalent disorder marked by excess water retention and substantial morbidity, motivating interest in the CLC-Ka chloride channel as a therapeutic target. Selectively inhibiting CLC-Ka without affecting the closely related CLC-Kb is essential for preventing serious side effects. However, developing isoform-selective inhibitors has been challenging because most small molecules do not distinguish between CLC-Ka and CLC-Kb, and the basis for selectivity in the few known exceptions remains unclear. The small molecule BIM1 preferentially inhibits CLC-Ka over CLC-Kb, providing an opportunity to dissect isoform-specific pharmacology. To investigate this mechanism, we determined cryo-EM structures of BIM1 and BIM15, a related nonselective analog, bound to a CLC-K variant engineered to match the human CLC-Ka binding pocket. Structural and computational analyses reveal that inhibition and isoform selectivity are anchored by interactions with a conserved lysine, with surrounding binding-site residues subtly tuning the local electrostatic environment to promote or disfavor these contacts. These analyses further identify a dynamic extracellular loop that intermittently occludes the access pathway, indicating its role as a gate for ions and inhibitors. BIM15 engages this gating loop more extensively than BIM1, suggesting that differential loop engagement contributes to inhibitor selectivity. To probe how gating reshapes this region, we solved the structure in the presence of Ca2+, which favors channel opening, and found the gating loop ordered and withdrawn from the pathway. Together, these findings elucidate how CLC-K channels gate and how subtle binding-site differences and loop dynamics shape isoform-specific drug binding, providing a foundation for designing next-generation CLC-Ka inhibitors.

  View details for DOI 10.64898/2026.02.17.706469

  View details for PubMedID 41756880

  View details for PubMedCentralID PMC12934935

• Cholesterol modulates the structural dynamics of the paddle motif loop of KvAP voltage sensor CURRENT RESEARCH IN STRUCTURAL BIOLOGY Das, A., Bysack, A., Raghuraman, H. 2024; 7: 100137

  Abstract

  KvAP is a prokaryotic Kv channel, which has been widely used as a model system to understand voltage- and lipid-dependent gating mechanisms. In phospholipid membranes, the KvAP-VSD adopts the activated/'Up' conformation, whereas the presence of non-phospholipids in membranes favours the structural transition to resting/'Down' state. The S3b-S4 paddle motif loop of KvAP-VSD is functionally important as this participates in protein-protein interactions and is the target for animal toxins. In this study, we have monitored the modulatory role of cholesterol - the physiologically-relevant non-phospholipid - on the organization and dynamics of the S3b-S4 loop of the isolated KvAP-VSD in membranes by site-directed fluorescence approaches using the environmental sensitivity of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD) fluorescence. Our results show that cholesterol alters the dynamic nature (rotational and hydration dynamics) of S3b-S4 loop in a segmental fashion, i.e., the residues 110 to 114 and 115 to 117 behave differently in the presence of cholesterol, which is accompanied by considerable change in conformational heterogeneity. Further, quantitative depth measurements using the parallax quenching method reveal that the sensor loop is located at the shallow interfacial region of cholesterol-containing membranes, suggesting that the sensor loop organization is not directly correlated with S4 helix movement. Our results clearly show that cholesterol-induced changes in bilayer properties may not be the predominant factor for the sensor loop's altered structural dynamics, but can be attributed to the conformational change of the KvAP-VSD in cholesterol-containing membranes. Overall, these results are relevant for gating mechanisms, particularly the lipid-dependent gating, of Kv channels in membranes.

  View details for DOI 10.1016/j.crstbi.2024.100137

  View details for Web of Science ID 001209680700001

  View details for PubMedID 38500801

  View details for PubMedCentralID PMC10945132

• Partial agonism in heteromeric GLUK2/GLUK5 kainate receptor PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Paudyal, N., Das, A., Carrillo, E., Berka, V., Jayaraman, V. 2025; 93 (1): 134-144

  Abstract

  Kainate receptors are a subtype of ionotropic glutamate receptors that form transmembrane channels upon binding glutamate. Here, we have investigated the mechanism of partial agonism in heteromeric GluK2/K5 receptors, where the GluK2 and GluK5 subunits have distinct agonist binding profiles. Using single-molecule Förster resonance energy transfer, we found that at the bi-lobed agonist-binding domain, the partial agonist AMPA-bound receptor occupied intermediate cleft closure conformational states at the GluK2 cleft, compared to the more open cleft conformations in apo form and more closed cleft conformations in the full agonist glutamate-bound form. In contrast, there is no significant difference in cleft closure states at the GluK5 agonist-binding domain between the partial agonist AMPA- and full agonist glutamate-bound states. Additionally, unlike the glutamate-bound state, the dimer interface at the agonist-binding domain is not decoupled in the AMPA-bound state. Our findings suggest that partial agonism observed with AMPA binding is mediated primarily due to differences in the GluK2 subunit, highlighting the distinct contributions of the subunits towards activation.

  View details for DOI 10.1002/prot.26565

  View details for Web of Science ID 001038275200001

  View details for PubMedID 37526035

  View details for PubMedCentralID PMC10830895

• Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores STAR PROTOCOLS Brahma, R., Das, A., Raghuraman, H. 2022; 3 (1): 101200

  Abstract

  Comprehensive understanding of a protein's function depends on having reliable, sophisticated tools to study protein structural dynamics in physiologically-relevant conditions. Here, we present an effective, robust step-by-step protocol to monitor the structural dynamics (including hydration dynamics) of a protein utilizing various site-directed fluorescence (SDFL) approaches. This protocol should be widely applicable for studying soluble proteins, intrinsically-disordered proteins, and membrane proteins. For complete details on the use and execution of this protocol, please refer to Das et al. (2020), Das and Raghuraman (2021), and Chatterjee et al. (2021).

  View details for DOI 10.1016/j.xpro.2022.101200

  View details for Web of Science ID 001141790100018

  View details for PubMedID 35252885

  View details for PubMedCentralID PMC8889417

• Conformational heterogeneity of the voltage sensor loop of KvAP in micelles and membranes: A fluorescence approach BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Das, A., Raghuraman, H. 2021; 1863 (5): 183568

  Abstract

  KvAP is a tetrameric voltage-gated potassium channel that is composed of a pore domain and a voltage-sensing domain (VSD). The VSD is crucial for sensing transmembrane potential and gating. At 0 mV, the VSD adopts an activated conformation in both n-octylglucoside (OG) micelles and phospholipid membranes. Importantly, gating-modifier toxins that bind at S3b-S4 loop of KvAP-VSD exhibit pronounced differences in binding affinity in these membrane-mimetic systems. However, the conformational heterogeneity of this functionally-important sensor loop in membrane mimetics is poorly understood, and is the focus of this work. In this paper, we establish, using intrinsic fluorescence of the uniquely positioned W70 in KvAP-VSD and environment-sensitive NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine) fluorescence of the labelled S3b-S4 loop, that the surface charge of the membrane does not significantly affect the topology and structural dynamics of the sensor loop in membranes. Importantly, the dynamic variability of the sensor loop is preserved in both zwitterionic (POPC) and anionic (POPC/POPG) membranes. Further, the lifetime distribution analysis for the NBD-labelled residues by maximum entropy method (MEM) demonstrates that, in contrast to micelles, the membrane environment not only reduces the relative discrete population of sensor loop conformations, but also broadens the lifetime distribution peaks. Overall, our results strongly suggest that the conformational heterogeneity of the sensor loop is significantly altered in membranes and this correlates well with its environmental heterogeneity. This constitutes the first report demonstrating that MEM-lifetime distribution could be a powerful tool to distinguish changes in conformational heterogeneity in potassium channels with similar architecture and topology.

  View details for DOI 10.1016/j.bbamem.2021.183568

  View details for Web of Science ID 000634801100001

  View details for PubMedID 33529577

• Effectiveness of dual-detergent strategy using Triton X-100 in membrane protein purification BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Das, A., Bysack, A., Raghuraman, H. 2021; 578: 122-128

  Abstract

  Membrane solubilization by detergents is a critical step for successful membrane protein purification. Alkyl maltoside detergents such as DDM and DM are very expensive and are commonly used to produce most of the high-quality proteins in stable and functional form. Recently, dual-detergent strategy using inexpensive detergents for membrane solubilization step has been shown to be highly effective in purifying different classes of membrane proteins in a cost-effective manner. In this work, we have monitored the effectiveness of 'dual-detergent strategy' towards successful purification of the isolated voltage sensing domain (VSD) of KvAP and the inward rectifying K+ channel, KirBac1.1. We demonstrate that the inexpensive detergent Triton X-100 extracts the activated conformation of the KvAP-VSD well without compromising the structural integrity of the sensor, and also retains its proper structural dynamics. Importantly, the cost associated with solubilizing the KvAP sensor can be reduced by ∼2000 fold. To the best of our knowledge, our results constitute the first report characterizing the purification of KvAP voltage sensor using an inexpensive detergent. However, the dual-detergent strategy using Triton X-100 for membrane solubilization is not effective for the purification of inward rectifying K+ channel, KirBac1.1 even in presence of high salt concentration during solubilization. We propose that the dual-detergent strategy will be useful for extracting stable and functional proteins that are both DDM- and DM-extractable, but will be ineffective if the protein is only DM-extractable. The relevance of the effectiveness of dual-detergent strategy with respect to the hydrophobic thickness of proteins is discussed.

  View details for DOI 10.1016/j.bbrc.2021.09.031

  View details for Web of Science ID 000701809800003

  View details for PubMedID 34560581

• Structural Dynamics of the Paddle Motif Loop in the Activated Conformation of KvAP Voltage Sensor BIOPHYSICAL JOURNAL Das, A., Chatterjee, S., Raghuraman, H. 2020; 118 (4): 873-884

  Abstract

  Voltage-dependent potassium (Kv) channels play a fundamental role in neuronal and cardiac excitability and are potential therapeutic targets. They assemble as tetramers with a centrally located pore domain surrounded by a voltage-sensing domain (VSD), which is critical for sensing transmembrane potential and subsequent gating. Although the sensor is supposed to be in "Up" conformation in both n-octylglucoside (OG) micelles and phospholipid membranes in the absence of membrane potential, toxins that bind VSD and modulate the gating behavior of Kv channels exhibit dramatic affinity differences in these membrane-mimetic systems. In this study, we have monitored the structural dynamics of the S3b-S4 loop of the paddle motif in activated conformation of KvAP-VSD by site-directed fluorescence approaches, using the environment-sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD). Emission maximum of NBD-labeled loop region of KvAP-VSD (residues 110-117) suggests a significant change in the polarity of local environment in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) membranes compared to OG micelles. This indicates that S3b-S4 loop residues might be partitioning to membrane interface, which is supported by an overall increased mean fluorescence lifetimes and significantly reduced water accessibility in membranes. Further, the magnitude of red edge excitation shift (REES) supports the presence of restricted/bound water molecules in the loop region of the VSD in micelles and membranes. Quantitative analysis of REES data using Gaussian probability distribution function clearly indicates that the sensor loop has fewer discrete equilibrium conformational states when reconstituted in membranes. Interestingly, this reduced molecular heterogeneity is consistent with the site-specific NBD polarization results, which suggest that the membrane environment offers a relaxed/dynamic organization for most of the S3b-S4 loop residues of the sensor. Overall, our results are relevant for understanding toxin-VSD interaction and gating mechanisms of Kv channels in membranes.

  View details for DOI 10.1016/j.bpj.2019.08.017

  View details for Web of Science ID 000515535500013

  View details for PubMedID 31547975

  View details for PubMedCentralID PMC7036696

• Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins FRONTIERS IN MOLECULAR BIOSCIENCES Raghuraman, H., Chatterjee, S., Das, A. 2019; 6: 96

  Abstract

  Membrane proteins mediate a number of cellular functions and are associated with several diseases and also play a crucial role in pathogenicity. Due to their importance in cellular structure and function, they are important drug targets for ~60% of drugs available in the market. Despite the technological advancement and recent successful outcomes in determining the high-resolution structural snapshot of membrane proteins, the mechanistic details underlining the complex functionalities of membrane proteins is least understood. This is largely due to lack of structural dynamics information pertaining to different functional states of membrane proteins in a membrane environment. Fluorescence spectroscopy is a widely used technique in the analysis of functionally-relevant structure and dynamics of membrane protein. This review is focused on various site-directed fluorescence (SDFL) approaches and their applications to explore structural information, conformational changes, hydration dynamics, and lipid-protein interactions of important classes of membrane proteins that include the pore-forming peptides/proteins, ion channels/transporters and G-protein coupled receptors.

  View details for DOI 10.3389/fmolb.2019.00096

  View details for Web of Science ID 000488120300001

  View details for PubMedID 31608290

  View details for PubMedCentralID PMC6774292

• Biochemical and biophysical characterization of a prokaryotic Mg<SUP>2+</SUP> ion channel: Implications for cost-effective purification of membrane proteins PROTEIN EXPRESSION AND PURIFICATION Chatterjee, S., Das, A., Raghuraman, H. 2019; 161: 8-16

  Abstract

  Although magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-β-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized 'dual-detergent strategy' to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3-14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3-14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of MgtE. Importantly, limited proteolysis assay, circular dichroism spectroscopy and ensemble tryptophan fluorescence strongly support the use of Triton X-100, in particular, as an inexpensive, alternative detergent for the purification of MgtE without compromising the structural integrity of the channel and Mg2+-induced gating-related conformational dynamics. Overall, these results are relevant for the cost-effective purification of stable and functional membrane proteins in general, and magnesium channels, in particular.

  View details for DOI 10.1016/j.pep.2019.04.005

  View details for Web of Science ID 000469892900002

  View details for PubMedID 31028884

  View details for PubMedCentralID PMC7116351