Rabindra Shivnaraine

All Publications

• Allosteric regulation and oligomerization of muscarinic cholinergic receptors Shivnaraine, R. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556701358

• Single-Molecule Analysis of the Supramolecular Organization of the M-2 Muscarinic Receptor and the G alpha(i1) Protein JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shivnaraine, R. V., Fernandes, D. D., Ji, H., Li, Y., Kelly, B., Zhang, Z., Han, Y. R., Huang, F., Sankar, K. S., Dubins, D. N., Rocheleau, J. V., Wells, J. W., Gradinaru, C. C. 2016; 138 (36): 11583-11598

  Abstract

  G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected by GTP?S. A complex of M2R and Gi1 was tetrameric in both, and activation by a full agonist plus GTP?S reduced the oligomeric size of Gi1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-G?i1 by the receptor-mimic mastoparan plus GTP?S, and constitutively active eGFP-G?i1 was predominantly dimeric. The oligomeric nature of Gi1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled G?i1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-Gi1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of Gi1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.

  View details for DOI 10.1021/jacs.6b04032

  View details for Web of Science ID 000383410700031

  View details for PubMedID 27494760

• Allosteric modulation in monomers and oligomers of a G protein-coupled receptor ELIFE Shivnaraine, R. V., Kelly, B., Sankar, K. S., Redka, D. S., Han, Y. R., Huang, F., Elmslie, G., Pinto, D., Li, Y., Rocheleau, J. V., Gradinaru, C. C., Ellis, J., Wells, J. W. 2016; 5

  Abstract

  The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.

  View details for DOI 10.7554/eLife.11685

  View details for Web of Science ID 000387459600001

  View details for PubMedID 27151542

  View details for PubMedCentralID PMC4900804