title: A modified infrared spectrometer with high time resolution and its application for investigating fast conformational changes of the GTPase Ras.
abstract: Time-resolved infrared spectroscopy is a valuable tool for the investigation of proteins and protein interactions. The investigation of many biological processes is possible by means of caged compounds, which set free biologically active substances upon light activation. Some caged compounds could provide sub-nanosecond time resolution, e.g., para-hydroxyphenacyl-guanosine 5'-triphosphate (GTP) forms GTP in picoseconds. However, the time resolution in single shot experiments with rapid-scan Fourier transform infrared (FT-IR) spectrometers is limited to about 10 ms. Here we use an infrared diode laser instead of the conventional globar and achieve a time resolution of 100 ns. This allows for the time-resolved measurement of the fast Ras(off) to Ras(on) conformational change at room temperature. We quantified the activation parameters for this reaction and found that the free energy of activation for this reaction is mainly enthalpic. Investigation of the same reaction in the presence of the Ras binding domain of the effector Raf (RafRBD) reveals a four orders of magnitude faster reaction, indicating that Ras·RafRBD complex formation directly induces the conformational change. Recent developments of broadly tunable quantum cascade lasers will further improve time resolution and usability of the setup. The reported 100 ns time resolution is the best achieved for a non-repetitive experiment so far.
authors.affiliation: Department of Biophysics, Ruhr-University Bochum, Universitäetsstr. 150, Bochum 44801, Germany.
authors.forename: Jie
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Biophysics, Ruhr-University Bochum, Universitäetsstr. 150, Bochum 44801, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.affiliation: Department of Biophysics, Ruhr-University Bochum, Universitäetsstr. 150, Bochum 44801, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
mesh_terms: D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000528:radiation effects; D054023:Lasers, Semiconductor; D008958:Models, Molecular; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000295:instrumentation* / Q000706:statistics & numerical data; D013696:Temperature; D013816:Thermodynamics; D013995:Time; D048490:raf Kinases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D048490:raf Kinases; D016283:Proto-Oncogene Proteins p21(ras)