Abstract
Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits
Highlights
Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein
We show that ChiT can be reconstituted with G-protein transducin (Gt)␥ subunits to form a functional heterotrimer that is amenable to structural analysis by high resolution NMR, thereby allowing us to probe differences in the conformation of G␣ in various states as well as the structural basis for signal transfer from R* to the heterotrimeric G-protein
Expression, Purification, and Heterotrimer Reconstitution of 15N-ChiT—Attempts to generate functional Gt␣ through inducible bacterial expression have not been successful due to insolubility of the expressed protein. This has led to the design and construction of numerous G␣ chimeras composed of various sequences from Gt␣ and Gi1␣, some of which are more amenable to soluble expression [29, 45]
Summary
Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. Most 1HN,15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP1⁄7AlF4؊/Mg2؉- and GTP␥S/Mg2؉-bound states Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of G␣ (“preactivation”) that may facilitate the interaction with R* and subsequent GDP/GTP exchange. Light triggers the cis 3 trans isomerization of the retinal chromophore to initiate structural changes in the transmembrane helices that results in the formation of the light-activated signaling state, metarhodopsin II or R* This is accompanied by small, yet functionally significant changes in the solvent-exposed cytoplasmic loops that lead to the formation of binding and activation sites for several signaling proteins, including Gt [2,3,4].
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