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A modified infrared spectrometer with high time resolution and its application for investigating fast conformational changes of the GTPase Ras.
- Source :
-
Applied spectroscopy [Appl Spectrosc] 2014; Vol. 68 (5), pp. 531-5. - Publication Year :
- 2014
-
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.
- Subjects :
- Binding Sites
Guanosine Triphosphate chemistry
Guanosine Triphosphate radiation effects
Lasers, Semiconductor
Models, Molecular
Photolysis
Protein Binding
Protein Conformation
Protein Interaction Mapping
Protein Structure, Tertiary
Proto-Oncogene Proteins p21(ras) metabolism
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Spectrophotometry, Infrared statistics & numerical data
Temperature
Thermodynamics
Time
raf Kinases metabolism
Guanosine Triphosphate analogs & derivatives
Proto-Oncogene Proteins p21(ras) chemistry
Spectrophotometry, Infrared instrumentation
raf Kinases chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1943-3530
- Volume :
- 68
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Applied spectroscopy
- Publication Type :
- Academic Journal
- Accession number :
- 25014595
- Full Text :
- https://doi.org/10.1366/13-07320