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Papers by Suparna Patowary 100449

Biophysical Journal, 2018

Recently, a couple of giga-seal automated patch clamp (APC) platforms in a standard 384-well plat... more Recently, a couple of giga-seal automated patch clamp (APC) platforms in a standard 384-well plate format have been introduced. These APC instruments can deliver higher throughput measurements with high-quality data for ion channel drug discovery. In this study, we developed an automated electrophysiology assay for the voltage-gated potassium channel, Kv1.3 as a test case on the Nanion SyncroPatch 384PE, one of the latest APC platforms. We achieved a high cell-catch rate (98% wells/plate, defined by membrane resistance >16 MU/cell) by screening plate types (number of holes and resistance), and obtained a stable seal (87% wells/plate, defined by initial/final seal resistance >500 MU/cell) by optimizing recording solutions. We generated data at an overall success rate (defined by initial peak current >300 pA/cell, initial/final seal resistance >500 MU/cell, and current stability <57% change/min) of 79% on average. Using the same platform, we validated a dose-response assay using clotrimazole, a potent Kv1.3 inhibitor. The assay was robust with Z' factor 0.52 and the success rate 75%. Altogether, our results demonstrated that the SyncroPatch represents a reliable platform for voltage-gated potassium channel assays.

Under the Supervision of Professor Valerică Raicu Recent advancements in fluorescence microscopy ... more Under the Supervision of Professor Valerică Raicu Recent advancements in fluorescence microscopy coupled with newly developed fluorescent tags have transformed Fluorescence (Förster) Resonance Energy Transfer (FRET) into a powerful tool studying in vivo molecular interactions with improved spatial (angstrom) resolution. Though widely used to study protein-protein interactions, generalizing and testing the FRET theory for oligomeric complexes containing multiple donors and acceptors has only become possible in recent years. Therefore, many aspects of it are yet unexplored. In this work, we tested the kinetic theory of FRET using linked fluorescent proteins located in the cytoplasm or at the plasma membrane. We used a novel method developed in our lab that combines an optical micro-spectroscope (OptiMiS) with a simple kinetic theory of FRET that relates the number and relative disposition of monomers within an oligomer to the measured FRET efficiency in terms of the pair-wise FRET efficiencies for an individual donor-acceptor pair in the oligomer. Using this iii framework, we showed that the measured FRET efficiencies of obligate trimers and tetramers in living cells are correctly predicted by the kinetic theory of FRET. The method was then used to study the oligomerization of G-protein coupled receptors (GPCRs), which are cell surface signaling proteins that constitute a large family of drug targets. The literature on GPCR homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. We used a novel FRET framework together with Optical Micro Spectroscopy (OptiMiS) technology and controlled expression of energy donor-tagged species of muscarinic M3 acetylcholine receptor, a GPCR of interest, to show that M3R exists as stable dimeric complexes at the plasma membrane, a large fraction of which interacts dynamically to form tetramers without the presence of trimers, pentamers, hexamers, etc. This was also supported by co-immunoprecipitation of receptors synthesized at distinct times. Based on these findings, we proposed a conceptual model that may reconcile the conflicting literature views on the quaternary structure of GPCRs.

Biophysical Journal, 2016

Biophysical Journal, 2015

Fö rster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy fr... more Fö rster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photoinsensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

SPIE Proceedings, 2011

Determination of the stoichiometry, structure, and distribution in living cells of protein comple... more Determination of the stoichiometry, structure, and distribution in living cells of protein complexes from analysis of single-molecular-complexes FRET. [Proceedings of SPIE 7903, 790324 (2011)]. Michael R. Stoneman, S. Patowary ...

Integrative biology : quantitative biosciences from nano to macro, 2013

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic ... more Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Förster Reso...

Biophysical Journal, 2012

G-protein-coupled receptors (GPCRs) such as human muscarinic acetylcholine receptor 3 (hM3) assum... more G-protein-coupled receptors (GPCRs) such as human muscarinic acetylcholine receptor 3 (hM3) assume dimeric/oligomeric forms in living cells while maintaining their ability to bind and activate G-proteins. The precise stoichiometry, quaternary organization, and stability of these receptor complexes in living cells remain a subject of significant controversy. We have investigated the organization of hM3 receptors in living cells using spectrally resolved two photon microscopy (SR-TPM). Wild type hM3 and a synthetic-ligand-regulated form of this receptor (RASSL), were expressed either constitutively or in an antibiotic dependent manner in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. A so-called donor of energy (Cerulean Fluorescent Protein) fused to RASSL can get excited by laser light and transfers its energy to nearby (<10nm) acceptors (Yellow Fluorescent Protein) fused to wild type hM3 receptors through a non-radiative process called Forster Resonance Energy Transfer (FRET). Using our SR-TPM microscope, we were able to collect images showing distributions of single complexes of hM3-RASSL and hM3-WT formed at various levels of RASSL receptor expression and determined their apparent efficiency of energy transfer (Eapp). The calculated Eapp for individual complexes were binned according to their values in order to obtain a cumulative histogram of Eapp for all complexes. The experimental data thus obtained were fitted to theoretical models [Raicu et al, Nature Photonics, 2009] in order to determine the quaternary structure of the M3 receptor, which turned out to be a rhombus-shaped tetramer.

Biophysical Journal, 2011

examined using fluorescence polarization microscopy. We characterized the domain organization of ... more examined using fluorescence polarization microscopy. We characterized the domain organization of the FG nups, which are required for transport of cargo through the NPC. This approach revealed both structured and unstructured domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. This technique allows the collection of structural information in vivo with the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nups, which are implicated in the mechanism of cargo transport.

Biophysical Journal, 2013

Biochemical Journal, 2013

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting ... more The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M3Rs (muscarinic M3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M3R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconci...