Abstract
Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg(2+) binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm(-1), respectively), α- and β-GDP (1214 and 1134/1103 cm(-1), respectively), and free phosphate (1078/991 cm(-1)). In contrast to Ras · GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated.
Highlights
Multiple turnover GTPase assays of G␣ are dominated by nucleotide exchange
We present here for the first time single turnover measurements of G␣i1 using time-resolved FTIR spectroscopy, an ultrasensitive method that can be applied in solution and has been successfully used for photoactivable proteins like bacteriorhodopsin [19, 20], channelrhodopsin [21], and other rhodopsins [22]
Multiple turnover GTPase assays like malachite green or radiometric phosphate tests using [␥-32P]GTP are widely used even though it is commonly known that GDP/GTP
Summary
Multiple turnover GTPase assays of G␣ are dominated by nucleotide exchange. Results: FTIR elucidates single turnover rates and individual phosphate vibrations. G␣ subunits are central molecular switches in cells They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of G␣i1. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg2؉ binding bias. Experiments were complemented by molecular dynamics simulations This broad approach provided detailed insights at atomic resolution and allows to identify key residues of G␣i1 in GTP hydrolysis and nucleotide exchange. The effect of nucleotide binding (G␣i1D272N) and Ras-like/all-␣ interface coordination (G␣i1D229N/G␣i1-D231N) on the nucleotide exchange reaction was elucidated
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