title: Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism.
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.
authors.affiliation: From the Biophysics Department, Ruhr-University Bochum, 44801 Bochum, Germany.
authors.forename: Grit
authors.initials: G
authors.lastname: Schröter
authors.affiliation: From the Biophysics Department, Ruhr-University Bochum, 44801 Bochum, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mann
authors.affiliation: From the Biophysics Department, Ruhr-University Bochum, 44801 Bochum, Germany carsten.koetting@rub.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.affiliation: From the Biophysics Department, Ruhr-University Bochum, 44801 Bochum, Germany klaus.gerwert@bph.rub.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C584114:GNAI1 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Fourier transform IR (FTIR); GTPase; enzyme mechanism; fluorescence; heterotrimeric G protein; molecular dynamics; mutagenesis; nucleotide exchange