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., <i>para</i>-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.

Temperature-sensitive gating of hCx26: high-resolution Raman spectroscopy sheds light on conformational changes

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Biomed. Opt. Express 5(7) 2054-2065 (2014)

High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events

D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, and P. M. W. French
Opt. Express 15(24) 15656-15673 (2007)

Far-infrared high-resolution Fourier transform spectrometer

Bruno Carli, Massimo Carlotti, Francesco Mencaraglia, and Enzo Rossi
Appl. Opt. 26(18) 3818-3822 (1987)

Thermospheric dynamics investigations with very high resolution spectrometers

G. Hernandez and R. G. Roble
Appl. Opt. 18(20) 3376-3385 (1979)

High-Resolution Grating Spectrometer for the Far Infrared

F. K. Kneubühl, J.-F. Moser, and H. Steffen
J. Opt. Soc. Am. 56(6) 760-764 (1966)