title: Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy.
abstract: Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gαi1, a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gαi1point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gαi1-T48A), γ-GTP (Gαi1-R178S), and for both β-GTP/γ-GTP (Gαi1-K46A, Gαi1-D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gαi1: we show that the GDP state of Gαi1carries a Mg2+, which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated.
authors.affiliation: Department of Biophysics, Ruhr University Bochum, Bochum, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mann
authors.affiliation: Westfälische Wilhelms-Universität Münster, Organisch-Chemisches Institut, Münster, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Höweler
authors.affiliation: Department of Biophysics, Ruhr University Bochum, Bochum, Germany. Electronic address: carsten.koetting@rub.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.affiliation: Department of Biophysics, Ruhr University Bochum, Bochum, Germany; CAS-MPG Partner Institute for Computational Biology (PICB) Shanghai, Shanghai, China. Electronic address: gerwert@bph.rub.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D020816:Amino Acid Motifs; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011789:Quantum Theory; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D006153:Guanosine Diphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D008274:Magnesium