Browsing by Author "Chatterjee, Sudeshna"

Now showing 1 - 4 of 4
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    Mechanistic Understanding of the Phosphorylation-Induced Conformational Rigidity at the AMPA Receptor C-terminal Domain
    (American Chemical Society, 2019) Chatterjee, Sudeshna; Dutta, Chayan; Carrejo, Nicole C.; Landes, Christy F.
    Phosphorylation at the intracellular C-terminal domain (CTD) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induces conformational rigidity. Such intracellular alterations to the AMPA receptor influence its functional responses, which are involved in multiple synaptic processes and neuronal signaling. The structure of the CTD still remains unresolved, which poses challenges toward providing a mechanism for the process of phosphorylation and deciphering the role of each phosphorylation step in causing the resultant conformational behavior. Herein, we utilize smFRET spectroscopy to understand the mechanism of phosphorylation, with the help of strategic point mutations that mimic phosphorylation. Our results reveal that first, phosphorylation at three target sites (S818, S831, and T840) is necessary for the change in the secondary structure of the existing disordered native sequence. Also, the results suggest that the formation of the tertiary structure through electrostatic interaction involving one specific phosphorylation site (S831) stabilizes the structure and renders conformational rigidity.
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    Stargazin Modulation of AMPA Receptors
    (Elsevier, 2016) Shaikh, Sana A.; Dolino, Drew M.; Lee, Garam; Chatterjee, Sudeshna; MacLean, David M.; Flatebo, Charlotte; Landes, Christy F.; Jayaraman, Vasanthi; Applied Physics Graduate Program
    Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of the receptor and how this distribution is affected by stargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.
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    The structure–energy landscape of NMDA receptor gating
    (Springer Nature, 2017) Dolino, Drew M.; Chatterjee, Sudeshna; MacLean, David M.; Flatebo, Charlotte; Bishop, Logan D.C.; Shaikh, Sana A.; Landes, Christy F.; Jayaraman, Vasanthi
    N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.
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    Understanding the activation-deactivation mechanism of glutamate receptor proteins at the single molecule level
    (2019-04-15) Chatterjee, Sudeshna; Landes, Christy F.
    Ionotropic glutamate receptor proteins form ion channels at the neuronal membrane and mediate excitatory synaptic neurotransmission in the mammalian central nervous system. These receptor proteins, such as NMDA and AMPA receptors, are structurally diverse and house multiple extracellular and intracellular binding sites, which can modulate activated (open) or deactivated (closed) states of the ion channel. The gating action (activation and deactivation) of the ion channels are controlled by a number of molecules (ligands) interacting with specific binding sites. Hence, these binding sites can serve as potential drug targets for controlling the aberrant activation of the ion channel that leads to a number of neurological disorders. In this research, we have utilized single molecule Förster Resonance Energy Transfer (smFRET) spectroscopy to provide a mechanistic understanding of the activation processes that the receptors undergo. We have probed, at a single molecule level, the structural changes explored by the NMDA receptor proteins, upon binding with several ligands that induce different modes of activation and have provided a comprehensive mechanism for their gating action. Furthermore, we have reported the conformational behavior induced by phosphorylation, at the structurally unresolved C-terminal domain of the AMPA receptors. We have demonstrated the capability of single molecule spectroscopy for studying such complex protein structures and deciphering mechanisms that would otherwise remain unresolved by ensemble measurements. The results from this study are expected to contribute to the ever-expanding research of targeted neuro-therapy.