Conformational changes at the carboxyl terminus of Galpha occur during G protein activation.
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.
Yang
C S
CS
Department of Physiology & Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Skiba
N P
NP
Mazzoni
M R
MR
Hamm
H E
HE
D017607:Aluminum Compounds / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007546:Isoquinolines / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents
D017607:Aluminum Compounds; D005456:Fluorescent Dyes; D007546:Isoquinolines; D011993:Recombinant Fusion Proteins; D012997:Solvents; C050992:tetrafluoroaluminate; C017475:lucifer yellow; D019204:GTP-Binding Proteins; D003545:Cysteine; D005459:Fluorides