Conformational changes at the carboxyl terminus of Galpha occur during G protein activation.

Journal Article (Journal Article)

To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Galpha were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Galphat/Galphai1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4--dependent increase in LY fluorescence (91%), indicating that Galpha activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4- before labeling led to a decreased incorporation of LY into Cys347 suggesting that Galpha activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Galpha and activated receptors, and may contribute to dissociation of activated Galpha subunit from activated receptor.

Full Text

Duke Authors

Cited Authors

  • Yang, CS; Skiba, NP; Mazzoni, MR; Hamm, HE

Published Date

  • January 22, 1999

Published In

Volume / Issue

  • 274 / 4

Start / End Page

  • 2379 - 2385

PubMed ID

  • 9891006

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.274.4.2379


  • eng

Conference Location

  • United States