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Structural Basis of Vertebrate Visio...

Gao, Yang.

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Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade./
Author:	Gao, Yang.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
Description:	167 p.
Notes:	Source: Dissertations Abstracts International, Volume: 80-03, Section: B.
Contained By:	Dissertations Abstracts International80-03B.
Subject:	Chemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10843841
ISBN:	9780438343245

Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade.
Gao, Yang.

Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 167 p.

Source: Dissertations Abstracts International, Volume: 80-03, Section: B.

Thesis (Ph.D.)--Cornell University, 2018.

This item must not be sold to any third party vendors.

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin, the GPCR, catalyzes the exchange of GDP for GTP on the heterotrimeric G protein transducin. This results in the dissociation of transducin into its component GTP-bound α subunit and the βγ subunit complex. Structural information for the rhodopsin-transducin complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. I have purified a stable detergent-solubilized complex between rhodopsin and transducin. The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol (LMNG) detergent, and purified with a combination of affinity and size exclusion chromatography. The complex is fully functional, with the stoichiometry of rhodopsin to transducin being 1:1. The molecular weight of the complex was calculated from small angle X-ray scattering (SAXS) data and is in good agreement with a model consisting of one rhodopsin molecule and one transducin molecule. The complex was visualized by negative-stain electron microscopy (EM), which revealed an overall architecture similar to that of the β2 adrenergic receptor-GS complex including a flexible helical domain in the transducin α subunit. The monodispersity, stability, and high yield of the purified complex allowed for further efforts toward obtaining a high-resolution structure of this important signaling complex. Recent X-ray and cryo-electron microscopy (cryo-EM) structures of complexes between different GPCRs and the stimulatory GS protein have revealed how these receptors converge structurally at the cytoplasmic end and engage the same GS protein, whereas, the cryo-EM structures of two GPCR-G i protein complexes show how the inhibitory Gi protein binds receptors in a different manner than the GS protein. In order to further our understanding of the mechanisms that underlie the specificity of GPCR-G protein interactions, and how they result in the G protein activation event, we determined a high-resolution cryo-EM structure of the light-activated, native bovine rhodopsin complexed with its cognate G protein partner transducin, which belongs to the inhibitory Gi family. The outward movement of transmembrane helix (TM) 6 and the rearrangement of TM5 at the intracellular side of Rho open up a binding cleft for the transducin α subunit, which engages rhodopsin in a different orientation than previously observed in other GPCR-GS protein complexes. The orientation of GT is also different from that of the Gi protein in the recently reported GPCR-Gi complexes, revealing the unexpected diversity regarding how GPCRs engage their G protein partners, even within the same G protein family. Moreover, the helical domain of the GαT subunit is less flexible than those resolved in previous GPCR-G protein complex structures and adopts an open conformation contacting the transducin β subunit, thus shedding new light on its involvement in the G protein activation event.

ISBN: 9780438343245Subjects--Topical Terms:

516420
Chemistry.

Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade.
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245 1 0 $a Structural Basis of Vertebrate Vision, a G Protein-Coupled Receptor Signaling Cascade.
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520 $a The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin, the GPCR, catalyzes the exchange of GDP for GTP on the heterotrimeric G protein transducin. This results in the dissociation of transducin into its component GTP-bound α subunit and the βγ subunit complex. Structural information for the rhodopsin-transducin complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. I have purified a stable detergent-solubilized complex between rhodopsin and transducin. The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol (LMNG) detergent, and purified with a combination of affinity and size exclusion chromatography. The complex is fully functional, with the stoichiometry of rhodopsin to transducin being 1:1. The molecular weight of the complex was calculated from small angle X-ray scattering (SAXS) data and is in good agreement with a model consisting of one rhodopsin molecule and one transducin molecule. The complex was visualized by negative-stain electron microscopy (EM), which revealed an overall architecture similar to that of the β2 adrenergic receptor-GS complex including a flexible helical domain in the transducin α subunit. The monodispersity, stability, and high yield of the purified complex allowed for further efforts toward obtaining a high-resolution structure of this important signaling complex. Recent X-ray and cryo-electron microscopy (cryo-EM) structures of complexes between different GPCRs and the stimulatory GS protein have revealed how these receptors converge structurally at the cytoplasmic end and engage the same GS protein, whereas, the cryo-EM structures of two GPCR-G i protein complexes show how the inhibitory Gi protein binds receptors in a different manner than the GS protein. In order to further our understanding of the mechanisms that underlie the specificity of GPCR-G protein interactions, and how they result in the G protein activation event, we determined a high-resolution cryo-EM structure of the light-activated, native bovine rhodopsin complexed with its cognate G protein partner transducin, which belongs to the inhibitory Gi family. The outward movement of transmembrane helix (TM) 6 and the rearrangement of TM5 at the intracellular side of Rho open up a binding cleft for the transducin α subunit, which engages rhodopsin in a different orientation than previously observed in other GPCR-GS protein complexes. The orientation of GT is also different from that of the Gi protein in the recently reported GPCR-Gi complexes, revealing the unexpected diversity regarding how GPCRs engage their G protein partners, even within the same G protein family. Moreover, the helical domain of the GαT subunit is less flexible than those resolved in previous GPCR-G protein complex structures and adopts an open conformation contacting the transducin β subunit, thus shedding new light on its involvement in the G protein activation event.
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