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

Publication ,  Journal Article
Yang, CS; Skiba, NP; Mazzoni, MR; Hamm, HE
Published in: J Biol Chem
January 22, 1999

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.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

January 22, 1999

Volume

274

Issue

4

Start / End Page

2379 / 2385

Location

United States

Related Subject Headings

  • Solvents
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Isoquinolines
  • GTP-Binding Proteins
  • Fluorides
  • Fluorescent Dyes
  • Cysteine
  • Chromatography, High Pressure Liquid
  • Biochemistry & Molecular Biology
 

Citation

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ICMJE
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Yang, C. S., Skiba, N. P., Mazzoni, M. R., & Hamm, H. E. (1999). Conformational changes at the carboxyl terminus of Galpha occur during G protein activation. J Biol Chem, 274(4), 2379–2385. https://doi.org/10.1074/jbc.274.4.2379
Yang, C. S., N. P. Skiba, M. R. Mazzoni, and H. E. Hamm. “Conformational changes at the carboxyl terminus of Galpha occur during G protein activation.J Biol Chem 274, no. 4 (January 22, 1999): 2379–85. https://doi.org/10.1074/jbc.274.4.2379.
Yang CS, Skiba NP, Mazzoni MR, Hamm HE. Conformational changes at the carboxyl terminus of Galpha occur during G protein activation. J Biol Chem. 1999 Jan 22;274(4):2379–85.
Yang, C. S., et al. “Conformational changes at the carboxyl terminus of Galpha occur during G protein activation.J Biol Chem, vol. 274, no. 4, Jan. 1999, pp. 2379–85. Pubmed, doi:10.1074/jbc.274.4.2379.
Yang CS, Skiba NP, Mazzoni MR, Hamm HE. Conformational changes at the carboxyl terminus of Galpha occur during G protein activation. J Biol Chem. 1999 Jan 22;274(4):2379–2385.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

January 22, 1999

Volume

274

Issue

4

Start / End Page

2379 / 2385

Location

United States

Related Subject Headings

  • Solvents
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Isoquinolines
  • GTP-Binding Proteins
  • Fluorides
  • Fluorescent Dyes
  • Cysteine
  • Chromatography, High Pressure Liquid
  • Biochemistry & Molecular Biology