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).

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) [15]. 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.

Fluorescence Resonant Energy Transfer (FRET)

FRET is an optical spectroscopic technique with which the dynamics of a single biomolecule can be followed (Fig. IV.i1). Incident optical light is absorbed by chromophore 1 at position 1 in the biomolecule. If sufficiently close by, i.e., if the protein is folded, the excitation energy can be transferred to a different chromophore 2, at position 2 in the molecule, resulting in the characteri-stic fluorescence signal of chromophore 2. If the inter-chromophore distance is too great, fluorescence will occur characteristic of chromophore 1. For single molecule FRET, the achievable time resolution is of the order of 10 ns [14]. Although a power ful technique for following the dynamic behavior of a single biomolecule, FRET cannot provide the detailed structural information available with the SwissFEL.