Time-resolved measurement techniques
Several methods of studying biochemical dynamics have been developed. Among the low-resolution methods are: optical absorption spectroscopy in the visible and IR regions, circular dichroism and Raman scattering. The hydrogen-deuterium exchange method, based on the different exchange rates of hydrogen isotopes for exposed and hidden amino acids in a protein immersed in D2O, detects folding or unfolding on the time scale of milliseconds or longer. The Fluoresence Resonant Energy Transfer (FRET) method allows a determination of the state of folding of a single protein molecule with a time resolution of the order of 10 ns [13, 14] (see Infobox).
Fig. IV.4. A pump-probe SAXS instrument, with time-resolved data
on the hemoglobin photocycle . The time resolution is given by
the 100 ps X-ray pulse.
Atomic scale information can be obtained by Nuclear Magnetic Resonance on the time scale of 10– 0.1 seconds, by the observation of peak splitting, and of ms – μs, by line broadening.
Sychrotron-based, time-resolved Small and Wide Angle X-ray Scattering (TR-SAXS/WAXS) from photo-triggered biomolecules in solution is capable of providing nm spatial resolution and 100 ps time-resolution, limited by the X-ray pulse length (see Fig. IV.4) . Biomolecules are carried by a liquid jet – at the SwissFEL this must be in vacuum – using the technology presented in Chapter II. An optical laser pulse excites the molecules, which are then probed, after a preset time delay, by the 100 ps X-ray pulse.
The same experimental station as used for time-resolved SAXS is also employed to per form pump-probe Laue crystallography. For the CO-photo-detachment in crystalline myoglobin, shown in Figure IV.5, broadband X-ray pulses (3% bandwidth, centered at 15 keV photon energy) were used. Each X-ray pulse had 1010 photons, 32 pulses were acquired for each crystal orientation, and 31 different orientations were measured, without laser pump and with various pump-probe delays. With stable, ultra-shor t pulses from the SwissFEL, such time-resolved pump-probe SAXS and Laue experiments will be possible with a much improved 20 fs time resolution. Optimally configured broad-band radiation for timeresolved Laue diffraction will be provided by detuning the individual undulator sections of the SwissFEL, at some cost in intensity.