Visualizing molecular structures in the course of a reaction process is one of the major grand challenges in chemistry, biology and physics. In particular, most chemical and biologically relevant reactions occur in solution, and solution-phase reactions exhibit rich chemistry due to the solute-solvent interplay. Studying photo-induced reactions in the solution phase offers opportunities for understanding fundamental molecular reaction dynamics and interplay between the solute and the solvent, but at the same time the interactions between solutes and solvents make this task challenging. Ultrafast emission, absorption and vibration spectroscopy in ultraviolet, visible and infrared regions have made possible the investigation of fast time-evolving processes. However, such time-resolved optical spectroscopic tools generally do not provide direct and detailed structural information such as bond lengths and angles of reaction intermediates because the spectroscopic signals utilizing light in the ultraviolet to infrared range cannot be directly translated into a molecular structure at the atomic level. In contrast, with the advance of X-ray synchrotron sources that can generate high-flux, ultrashort X-ray pulses, time-resolved X-ray diffraction (scattering) and absorption techniques have become general and powerful tools to explore structural dynamics of matters. Accordingly, the techniques have been successfully applied to studying various dynamics of chemical and biological systems (Coppens, 2003; Coppens et al., 2004; Ihee, 2009; Ihee et al., 2005b; Kim et al., 2002; Schotte et al., 2003; Srajer et al., 1996; Techert et al., 2001; Tomita et al., 2009) and of condensed matters (Cavalieri et al., 2005; Cavalleri et al., 2006; Collet et al., 2003; Fritz et al., 2007; Gaffney et al., 2005; Lee et al., 2005; Lindenberg et al., 2005). On one hand, time-resolved X-ray diffraction enables us to access to the mechanism of structural transformations at the atomic level in crystalline state (Collet et al., 2003; Schotte et al., 2003; Srajer et al., 1996; Techert et al., 2001). On the other hand, time-resolved X-ray absorption fine structure (XAFS) (Chen et al., 2001; Saes et al., 2003; Sato et al., 2009) and time-resolved solution scattering (Davidsson et al., 2005; Ihee, 2009; Ihee et al., 2005a; Plech et al., 2004) can probe structural dynamics in non-crystalline states of materials, complementing the X-ray diffraction technique.
In particular, time-resolved X-ray liquidography (TRXL), which is also known as time-resolved X-ray solution scattering (TRXSS), provides rather direct information of transient molecular structures because scattering signals are sensitive to all chemical species present in the sample and can be compared with the theoretical scattering signal calculated from three-dimensional atomic coordinates of involved chemical species. Accordingly, time-resolved X-ray liquidography using 100-picosecond X-ray pulses from a synchrotron source has been effective in elucidating molecular geometries involved in photoinduced reaction pathways, elegantly complementing ultrafast optical spectroscopy (Cammarata et al., 2008; Christensen et al., 2009; Davidsson et al., 2005; Georgiou et al., 2006; Haldrup et al., 2009; Ichiyanagi et al., 2009; Ihee, 2009; Ihee et al., 2005a; Kim et al., 2006; Kong et al., 2008 2008,Lee et al., 2008b; Plech et al., 2004; Vincent et al., 2009, Wulff et al., 2006).
Time-resolved X-ray liquidography has been developed by combining the pulsed nature of synchrotron radiation and of lasers. In a typical experiment, a reaction is initiated by an ultrashort optical laser pulse (pump), and the time evolution of the induced structural changes is probed by the diffraction of a time-delayed, short X-ray pulse as a function of the time delay between the laser and X-ray pulses. In other words, the X-ray pulse replaces the optical probe pulse used in time-resolved optical pump-probe spectroscopy. X-ray pulses with a temporal duration of 50 ~ 150 ps are generated by placing an undulator in the path of electron bunches in a synchrotron storage ring.
In this chapter, we aim to review the experimental details and recent applications of time-resolved X-ray liquidography. Especially, we describe the details of the TRXL setup in NW14A beamline at KEK, where polychromatic X-ray pulses with an energy bandwidth of ΔE/E ~ 1 – 5% are generated by reflecting white X-ray pulses (ΔE/E = 15%) through multilayer optics made of W/B4C or depth-graded Ru/C on silicon substrates. Unlike in conventional X-ray scattering/diffraction experiments, where monochromatic X-rays are used to achieve high structural resolution, polychromatic X-ray pulses containing more photons than monochromatic X-ray pulses are used at the expense of the structural resolution because a higher signal-to-noise ratio is desirable in the TRXL experiment. In addition, we describe in detail the principle of synchronization between the laser and synchrotron X-ray pulses, which is one of the key technical components needed for the success of time-resolved X-ray experiments, and has been vigorously implemented in well-established experimental techniques using synchrotron radiation, such as diffraction, scattering, absorption and imaging. Finally, some examples of applications to various reaction systems ranging from small molecules to proteins are described as well.
2.1. Optical-pump and X-ray-scatter scheme
In a typical TRXL experiment, an ultrashort optical laser pulse initiates photochemistry of a molecule of interest in the solution phase, and an ultrashort x-ray pulse from a synchrotron facility, instead of an ultrashort optical pulse used in the optical pump-probe experiment, is sent to the reacting volume to probe the structural dynamics inscribed on the time-resolved x-ray diffraction signals as a function of reaction time. TRXL data have been collected using an optical-pump and x-ray-probe diffractometer in the beamline ID09B at ESRF (Bourgeois et al., 1996; Wulff et al., 1997) and the beamline NW14A of PF-AR at KEK (Nozawa et al., 2007). The beamline 14IDB at APS also has the capability of collecting TRXL data. The experimental setup is schematically illustrated in Fig. 1. It comprises a closed capillary jet or open-liquid jet to supply the solution that are pumped by laser pulses and scatter X-rays, a pulsed laser system to excite the sample, a pulsed synchrotron source to produce ultrashort X-ray pulses to scatter from the sample, a synchronized high-speed chopper that selects single X-ray pulses, and an integrating charge-coupled device (CCD) area detector.
2.2. Pulsed nature of synchrotron radiation
Synchrotron radiation is described as the radiation from charged particles accelerated at relativistic velocities by classical relativistic electrodynamics. It provides excellent characteristics as an X-ray source such as small divergence, short wavelength, linear or circular polarization, etc. Synchrotron radiation has another useful feature for time-resolved X-ray technique, short-pulsed nature, due to the periodic acceleration of charged particles in storage ring. Electrons circulating in storage ring irradiate synchrotron radiation and lose their energy. In order to compensate for the energy loss, a radio frequency (RF) oscillator accelerates electrons periodically at a harmonic frequency of the revolution frequency
2.3. X-ray source characteristics and isolation of a single X-ray pulse
Synchrotron radiation is operated at MHz to GHz repetition rate depending on the bunch-filling modes of the storage ring. In particular, time-resolved experiments at synchrotron radiation facilities primarily require sparse bunch-filling mode of the storage ring operation such as single-bunch or hybrid modes. In general, X-ray detectors have a relatively slow response time and, furthermore, two-dimensional X-ray area detectors (e.g. CCD) have no fast gating capabilities. Due to such limitation of X-ray detectors, isolation of a single X-ray pulse from a pulse train is crucial for the success of time-resolved X-ray experiments. Since a single pulse can be readily isolated by using a fast chopper in sparse bunch-filling mode, the operation in the single-bunch or hybrid mode is highly desirable for time-resolved X-ray experiments.
The 6.5 GeV PF-AR is fully operated in a single-bunch mode for about 5000 hours/year. Electrons with a ring current of 60 mA (75.5 nC per bunch) are stored in a single electron bunch with a life time of around 20 hours. The RF frequency and harmonic number of the PF-AR are 508.58 MHz and 640, respectively. Therefore, the X-ray pulses are delivered at a frequency of 794 kHz (= 508.58 MHz / 640) with a pulse duration of about 60 ps (rms). A schematic drawing of the beam line NW14A is shown in Fig. 2.
The beam line has two undulators with a period length of 20 mm (U20) and 36 mm (U36). The U20 gives the 1st harmonic in the energy range of 13–18 keV. The energy bandwidth of the 1st harmonic is ΔE/E = 15%, which is utilized as a narrow-bandwidth white beam for TRXL experiments. The U36 covers an energy range of 5–20 keV with 1st, 3rd, and 5th harmonics, and useful for X-ray spectroscopy experiments. The measured photon flux from U36 and U20 at several gaps is shown in Fig. 3.
In order to isolate a single X-ray pulse from the sources, double X-ray choppers are equipped at the NW14A. The first chopper, called as heat-load chopper, has an opening time of 15 μs and is used to isolate 10-pulse train at 945 Hz (Gembicky et al., 2007). The second X-ray chopper, made by Forschungszentrum Jülich (Lindenau et al., 2004), consists of a rotor furnished with a narrow channel for the beam passage and isolates a single X-ray pulse from the 10-pulse train. The Jülich chopper realizes continuous phase locking with timing jitter less than 2 ns. The opening time of the channel at the center of the tapered aperture is 1.64 μs. If the repetition frequency of the pump-probe experiment is lower than 945 Hz, as is the case of using 10-Hz YAG laser system, a millisecond X-ray shutter (UNIBLITZ, XRS1S2P0) is set up between the X-ray chopper and the sample.
2.4. Energy bandwidth of the incident X-ray beam
In order to gain maximum X-ray photon flux at 1 kHz repetition rate, energy bandwidth of the incident X-ray is the key issue. The X-ray pulse with 3% energy bandwidth of the first harmonics of the undulator has been used for TRXL experiments in the beamline ID09B at ESRF (Cammarata, 2008; Christensen et al., 2009; Davidsson, 2005; Georgiou, 2006; Ichiyanagi, 2009; Ihee, 2009; Ihee, 2005a; Kim, 2006; Kong, 2008, 2008 Lee, 2008b; Plech, 2004; Vincent, 2009; Wulff, 2006). For example, the structural dynamics of C2H4I2 in methanol were studied at the ID09B beamline (Ihee, 2005a), and the reaction pathways and associated transient molecular structures in solution were resolved by the combination of theoretical calculations and global fitting analysis.
On the other hand, high-flux white X-ray at NW14A has ΔE/E = 15% energy bandwidth when the undulator U20 is used due to relatively large electron beam emittance of PF-AR. In order to examine the feasibility of time-resolved liquidography with such a large bandwidth and to search for the optimal bandwidth, we simulated the Debye scattering curves for the reaction C2H4I2 → C2H4I + I using (i) a 15% bandwidth with the default X-ray energy distribution, such as the undulator spectrum at the NW14A beamline, (ii) a Gaussian spectrum with a 5% bandwidth, (iii) a Gaussian spectrum with a 1% bandwidth, and (iv) a Gaussian spectrum with a 0.01% energy bandwidth, as shown in Fig. 4.
Although the photon flux of X-ray pulse increases with the energy bandwidth of the X-ray, the simulation shows that the default X-ray spectrum that has a 15% energy bandwidth as well as a long tail is not suitable for the time-resolved liquidography experiment owing to deteriorated structural resolution. Especially, the long tail of the default X-ray spectrum further blurs the scattering pattern at high scattering angles than when a symmetric Gaussian spectrum of the same bandwidth is assumed. As a result of the asymmetric lineshape, the X-ray spectrum with a long tail at ID09B of ESRF with a 3% bandwidth is effectively comparable to a symmetric Gaussian spectrum with a 10% bandwidth. In contrast, the scattering curve calculated from the Gaussian spectrum with a 5% energy bandwidth is similar in its structural resolution to that obtained from a 0.01% energy bandwidth (monochromatic) Gaussian spectrum. Furthermore, the total flux of the 5% energy bandwidth X-ray beam is higher than that of the monochromatic X-ray (a 0.01% energy bandwidth) generated from a Si single crystal by a factor of 500. These estimations clearly suggest that the X-ray pulses with ΔE/E of 5% is appropriate for time-resolved X-ray liquidography experiment since it can provide a strong scattering signal without much sacrificing the structural resolution. Thus, we reduced the bandwidth of the X-ray pulses from the default 15% to less than the 5 % energy bandwidth.
The multilayer optics produces X-ray pulses with a 1% to 5% energy bandwidth and allows us to measure TRXL with the undulator at the NW14A beamline. We used two types of multilayer optics. The first optics, made of W/B4C (d =27.7 Å, X-ray Company, Russia) on a Si single crystal with a size of 50×50×5 mm3, provides an X-ray spectrum with a 1% energy bandwidth, as shown in Fig. 5(a). The peak energy of the X-ray spectrum can be changed by tilting the angle of the multilayer optics. The second multilayer optics, which is made of depth-graded Ru/C layer (average d = 40 Å, NTT Advanced Technology, Japan), produces a 5% energy bandwidth X-ray spectrum, as shown in Fig. 5(b). A white X-ray with a photon flux of 1 × 109 photons/pulse is produced at a 1 kHz repetition rate. When multilayer optics with 1% and 5% energy bandwidths are used at the downstream of the Jülich chopper, the photon flux of 6 × 107 and 3 × 108 photons/pulse is obtained, respectively.
2.5. Synchronization of laser and X-ray pulses
NW14A is equipped with a 150-fs Ti:sapphire regenerative amplifier laser system (Spectra Physics, Millenia, Tsunami, Spitfire, Empower). The Ti:sapphire laser system produces optical pulses at 800 nm at a 945-Hz repetition rate, with the pulse energy reaching up to 800 μJ/pulse. The laser is installed in a laser booth next to the experimental hutch. An optical parametric amplifier (Light Conversion, TOPAS-C) is also installed in the laser booth for conversion of 800 nm light to broad spectral range from visible to mid-infrared region. The laser beam is brought to the sample in the experimental hutch through a beam duct for the laser. The synchronization of X-ray and laser pulses is based on the RF master clock, by which an electron bunch is driven in the storage ring. When the X-ray experiment is conducted with a 945 Hz Ti:sapphire-laser and a detector that has no gating capabilities (e.g. CCD), an X-ray chopper is required to synchronize the X-ray and laser pulses at a 1:1 ratio. The timing chart of the synchronization is shown in Fig. 6.
The X-ray pulse is emitted every 1.26 μs (794 kHz = 508 MHz / 640) from the PF-AR. After the RF amplifier, the RF master clock signal of PF-AR is split into two major timing components: one for the laser system and the other for the X-ray chopper system. In the X-ray chopper system, the 508 MHz RF and the 794 kHz revolution signals are used as the clock and the reference signals, respectively. A 945 Hz (794 kHz / 840) repetition frequency of the X-ray pulses is then selected to trigger the Ti:sapphire 150-fs laser system running at the same repetition frequency. In the laser system, the mode-locked Ti:sapphire oscillator operating at 85 MHz (508 MHz / 6) synchronized with the X-ray pulses provides seed pulses to the regenerative amplifier. The seed pulses trigger the regenerative amplifier pumped by the Q-switched Nd:YLF laser at 945 Hz (85 MHz / 89600). Then, 945 Hz laser pulses are directed to the sample position by a series of mirrors. The pulse trains of pumping laser and probing X-ray pulses at the sample are shown together in Fig. 6. The timing of the delay between the two pulse trains is controlled by changing the ejection timing of the laser pulses from the regenerative amplifier using a phase shifter (Candox). The timing of the X-ray and the laser is measured with an InGaAs metal-semiconductor-metal (MSM) photodetector (Hamamatsu, G7096) coupled to a high-frequency preamplifier and a 2.5 GHz digital oscilloscope (Tektronix, DPO7254). The rise time of the MSM photodetector is typically 40 ps, which is faster than the X-ray pulse duration, and the photodetector is set at the sample position.
2.6. Spatial and temporal overlaps
In order to increase the signal-to-noise ratio of the TRXL data and define accurate time delay between laser and X-ray pulses, the laser and X-ray pulses have to be overlapped at the sample both spatially and temporally. To check the temporal overlap, we place a fast InGaAs detector at the sample position and record the time traces of the laser and X-ray pulses along a single time axis monitored by a 2.5 GHz digital oscilloscope. By adjusting the laser firing time, it is possible to adjust the relative timing between the two pulses within a few picoseconds. During an experiment, the time traces of the laser and X-ray pulses are monitored by fast photodiodes simultaneously and non-intrusively.
The spatial overlap between X-ray and laser pulses is achieved by using a 50 μm diameter pinhole placed at the sample position. The pinhole is located at the center of X-ray beam, and then the laser beam is moved across the pinhole by scanning the position of the focusing lens until it passes through the center of the pinhole. To ensure precise spatial overlap, we monitor the intensity of scattering induced by thermal expansion in a liquid solvent, which typically occurs in 1 μs with our beam sizes. Specifically, the ratio of scattered intensities in the inner and outer disks of the solvent signal is monitored. Once the sample expands, the solvent signal shifts to lower scattering angles, leading to the increase of low-angle scattering and the decrease of high-angle scattering. Therefore, the ratio between the inner and outer part of the solvent signal changes in proportion with the laser excitation. The X-ray beam is typically vertically 200 μm and horizontally 250 μm. The laser spot is of circular shape with a diameter of 300 – 400 μm.
2.7. Sample environment and data acquisition
Two different types of sample cell systems have been used: a diluted solution of 0.5~100 mM concentration or pure solvent is prepared and circulated through either a capillary or through an open-jet sapphire nozzle. Such flow systems provide a stable liquid flow of ~0.3 mm thickness at a speed ensuring the refreshment of probe volume for every laser pulse (typically ~3 m/s). In the capillary-based system, the solution is flowed through a quartz capillary of 0.3 mm diameter. In the open-jet system, the capillary is removed and the solution is passed between two flat sapphire crystals with a spacing of 0.3 mm (Kyburz), which produces a stable naked liquid sheet directly exposed to the laser/x-ray beams. The open-jet system producing a bare liquid jet has the advantage over the closed capillary system in terms that the scattering background arising from the glass material of the capillary is eliminated and thus the signal-to-noise-ratio substantially improves. The lower background also helps to enhance the accuracy of the normalization process. In addition, the capillary jet often encounters a problem that the excitation laser drills a hole in the capillary.
The molecules in the jet are excited by laser pulses from the femtosecond laser system described above. To maximize the population of transients and photoproducts, the laser pulse energy (typically 25 – 100 μJ depending on the excitation wavelength) is set to be relatively higher than that used in typical time-resolved optical spectroscopy, and thus multi-photon excitation often occurs. In general, one wants to follow photochemistry induced by only one-photon absorption that the laser pulse duration of ~100 fs is stretched to ~2 ps by introducing chirp from a pair of fused-silica prisms inserted before the sample. To probe slow photoinduced dynamics, a nanosecond laser system is used instead of the femtosecond laser system.
The laser beam is generally directed to the sample with a 10 degree tilt angle relative to the X-ray beam. The scattered X-ray diffraction signal is recorded by an area detector (MarCCD165, Rayonics, 2048 × 2048, ~80 μm effective pixel size) with a sample-to-detector distance of ~45 mm. A typical exposure time is ~5 s, and, given the ~1 kHz repetition rate of the laser/X-ray pulses, the detector receives 5 × 103 X-ray pulses and ~5 × 1012 X-ray photons per image. Diffraction data are collected for typically 10 or more time-delays (
3. Data processing and analysis
3.1. Conversion of 2D images into 1D curves
The two-dimensional diffraction images are radially integrated into one-dimensional intensity curve,
where the constant α is a damping constant to account for the finite experimental q range. In principle, the errors in the r-space can be also obtained from the same procedure as the one described for the q-space data: The sine-Fourier transform of every single qΔS(q,t) is taken and then averaged over all rΔR(r,t) curves, which defines a meaningful standard deviation.
3.2. Data analysis
We fit the experimental difference intensities (ΔS(q,t)
where R is the ratio of the number of solvent molecules to that of solute molecules, k is the index of the solute (reactants, intermediates and products), c
The basic strategy of the least square fits to the experimental data is to minimize the total χ2 iteratively in a global fitting procedure, simultaneously minimizing the differences between the experimental and theoretical curves at all positive time delays. The definition of chi-square (χ2) used is as follows.
The polychromacity of the X-ray beam has to be taken into account when a ΔS(q,t)
The fitting parameters include the rate coefficients, the fraction of the excited molecules, the fraction of the molecules undergoing structural changes, and the laser beam size. Structural parameters such as bond lengths and angles and energy levels of chemical species can be included as fitting parameters.
3.3. Example: Photochemistry of CHI3
Fig. 8A shows a comparison of qΔS(q, t)
4.1. On the issue of isomer formation from CHI3 in methanol
TRXL has been used to capture the molecular structures of intermediates and their reaction kinetics for various photochemical processes. In the following, we present some application examples ranging from small molecules to proteins, which illustrate the wide applicability of TRXL.
The first example is the photochemistry of iodoform (CHI3). According to previous time-resolved spectroscopic studies (Wall et al., 2003; Zheng et al., 2000), the CHI2 radical and I atom generated upon excitation at 267 nm geminately recombine to form iso-iodoform within the solvent cage as the main species (quantum yield of at least 0.5) with a rise time of 7 ps and this iso-iodoform survives for up to microseconds. To investigate the possibility of the isomer formation, we performed the global fitting analysis on the TRXL data with two candidate reaction pathways ([CHI3 → CHI2 + I; simple dissociation channel] and [CHI3 → CHI2-I; isomer formation channel]). As shown in Fig. 8, the isomer channel reaction model is not compatible with the TRXL data, but a simple dissociation channel gives good agreement
(Lee et al., 2008a). Furthermore, when both reaction models are included in the fit, the fraction of the isomer-formation process converges to zero, confirming that the iso-iodoform should be a minor species if it forms at all. Since the X-ray pulse width used in this study is ~100 ps (fwhm), the formation of iso-iodoform as a major species on time scales shorter than our experimental time resolution cannot be ruled out. The subsequent kinetics obtained from TRXL was detailed in the previous section (Data Analysis). It should be noted that the data show that the formation of I2 is dominant over other possible recombination products such as CHI3 (from CH3 and I) and C2H6 (from two CH3).
4.2. Protein folding of cytochrome c
Protein structural changes in solution have been mainly characterized by time-resolved optical spectroscopic methods that, despite their high time resolution (<100 fs), are only indirectly related to three-dimensional structures in space. For protein crystals, a combination of high time resolution and structural sensitivity has become readily available with the advent of sub-nanosecond Laue crystallography (Ihee et al., 2005b; Moffat, 2001; Schotte et al., 2003; Srajer et al., 1996), but its applicability has been limited to a few model systems due to the stringent prerequisites such as highly-ordered and radiation-resistant single crystals. More importantly, crystal packing constraints might hinder biologically relevant motions. Owing to such limitations, the time-resolved X-ray crystallography has been applied to only reversible reactions in single crystals, and it cannot be simply used to study irreversible reactions such as protein folding. To obtain information about protein motions in a more natural environment, X-ray scattering and nuclear magnetic resonance (NMR) methods have been mainly used as direct structural probes of protein structures in solution (Grishaev et al., 2005; Schwieters et al., 2003). Due to the inverse relationship between the interatomic distance and the scattering angle, the scattering from macromolecules is radiated at smaller scattering angles and is typically called as small-angle X-ray scattering (SAXS) or wide-angle X-ray scattering (WAXS) for scattering angles larger than conventional SAXS angles. The SAXS is sensitive to overall structure, for example, overall size and shape, of the protein, while wide-angle X-ray scattering (WAXS) gives more detailed information on the tertiary and quaternary structure such as the fold of helices and sheets. However, thus far, the time resolution had been limited to 160 μs at best (Akiyama et al., 2002). As well, NMR is a powerful technique for structure determination in solution, but it works best for small proteins and needs properly labeled samples (Kainosho et al., 2006). More importantly, due to the nature of microwave pulses, the time resolution of protein NMR is inherently limited to milliseconds.
In case of protein solutions, the relatively low concentration (only a few mM or less) make TRXL measurements non-trivial, and the large molecular size of proteins (more than thousand times larger than small molecules) complicates the structural analysis. However, recent TRXL data from model proteins in solution have demonstrated that the medium to large-scale dynamics of proteins is rich in information on time scales from nanoseconds to milliseconds (Cammarata et al., 2008). TRXL methodology has been applied to human haemoglobin (Hb), a tetrameric protein made of two identical αβ dimers, that is known to have at least two different quaternary structures (a ligated stable “relaxed” (R) state and an unligated stable “tense” (T) structures) in solution. The tertiary and quaternary conformational changes of human hemoglobin triggered by laser induced ligand dissociation have been identified using the TRXL method. A preliminary analysis by the allosteric kinetic model gives a time scale for the R-T transition of ~1–3 μs, which is shorter than the time scale derived with time-resolved optical spectroscopy. The optically induced tertiary relaxation of myoglobin (Mb) and refolding of cytochrome c (Cyt-c) have been also studied with TRXL. As previously mentioned, the advantage of TRXL over time-resolved X-ray protein crystallography is that it can probe irreversible reactions as illustrated with the folding of cytochrome c as well as reversible reactions such as ligand reactions in heme proteins.
The basic idea of protein folding is that the three-dimensional structure of proteins is mainly determined by their amino acid sequences. Unfolded polypeptide chains use this information to accurately and quickly fold into their native structures (Fig. 10a). The optically triggered folding of horse heart Cyt-c has been extensively studied with spectroscopic techniques (Chen et al., 1998; Jones et al., 1993) and also by using fast-mixing SAXS (Akiyama et al., 2002). Cyt-c is a single domain protein similar to Mb. Unlike Hb and Mb, Cyt-c does not usually bind external ligands such as CO since the iron atom of the heme group is covalently coordinated to the Met-80 residue of the protein. However, if Cyt-c is partially unfolded with a denaturing agent, it is possible to replace the Met-80 residue with CO and the CO ligand can be optically dissociated, thereby initiating the re-folding process. The time-dependent evolution of the TR-WAXS signal of Cyt-c after photolysis is evident, especially in the small-angle region (Fig. 10b). As a preliminary analysis, we fitted the observed signal as a linear combination of one pattern at the earliest time delay, 32 μs, and
the other at the latest time delay, 0.2 s. This simple approach reproduces the experimental data at all times very well. The plot of the weighting factor of the late time component against time is shown in Fig. 10c and a simple exponential analysis yields a time scale of about 25 ms for the CO-photolysis-triggered folding.
5. Summary and future perspectives
In this chapter, we have described the principle and experimental details of TRXL technique with recent examples of its applications. With 100-ps X-ray pulses readily available from synchrotron radiation, TRXL has been established as a powerful tool for characterizing fast structural transition dynamics of chemical reactions and biological processes, ranging from small molecules to proteins in solution. In particular, the technique provides rather direct information on transient molecular structures since scattering signals are sensitive to all chemical species present in the sample unlike in the optical spectroscopy. Although there still remain challenges to overcome, for example, limited structural and time resolution, TRXL is expected to play an important role in revealing transient structural dynamics in many other systems in solution and liquid phases, especially with the aid of next-generation X-ray sources. At the frontier of the technical advances supporting such bright prospects of TRXL is the advent of linac-based X-ray light sources, which can generate X-ray pulses of femtosecond duration. They include self-amplified spontaneous emission X-ray free electron lasers (SASE-XFEL) and energy recovery linacs (ERL) that are currently under development will be available in the near future.
Among these novel X-ray sources, the high-gain XFEL using SASE promises to generate highly coherent, femtosecond X-ray pulses on the order of 100 fs with a high photon flux up to 1013 photons per pulse. The superb time resolution of XFEL will enable us to access reaction dynamics in femtosecond time regime, elucidating much more details of ultrafast structural dynamics. Also, the high flux of XFEL provides the potential for single-shot collection of the XFEL signal. On the other hand, ERL can be operated at a high repetition rate on the order of MHz to GHz. Such high repetition rate capability of ERL will be able to significantly improve the signal-to-noise ratio of TRXL signal since TRXL is basically a perturbative, pump-probe type experiment. With such a high-repetition rate X-ray source, TRXL can be implemented combined with a high-repetition rate oscillator instead of femtosecond amplified lasers, which is commonly operated at only a kHz rate. Furthermore, the nanometer-scale size of the X-ray beam from the ERL (typically 100-nm diameter) will allow tight focusing of the laser beam down to the order of micrometers, enabling the collection of signal from a small volume of sample. Since the scattering signal from the small area will be relatively weak, low-noise and fast-gatable two-dimensional detectors are desirable for future ERL-applied TRXL experiments. The development of pixel detectors using silicon-on-insulator technology will pave the way for such high-performance two-dimensional detectors.
The excellent beam characteristics of the ERL will be further extended to develop the coherent X-ray source, for example, oscillator-type XFEL (XFEL oscillator or XFEL-O) (Kim et al., 2008). The X-ray source generating fully coherent X-ray pulses will serve as the ultimate X-ray light source with superb spatial and temporal coherence. Then, what kind of potential applications can we expect once fully coherent X-ray pulses become available? For example, by making an analogy to the ultrafast optical spectroscopy that fully takes advantage of the temporal coherence of ultrashort optical laser pulses, one could imagine phase-coherent spectroscopy in the X-ray regime with controlled timing, phase, and intensity among multiple, coherent X-ray pulses (Mukamel et al., 2009). As X-ray radiation has the sub-nm wavelength, which corresponds to the sub-attosecond period in the time domain, X-ray pulses offer much higher spatial and temporal resolution than achievable in the optical regime. Thus, the development of X-ray sources that can generate coherent X-ray pulses will revolutionize the whole X-ray science.
Akiyama S. Takahashi S. Kimura T. Ishimori K. Morishima I. Nishikawa Y. Fujisawa T. 2002Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering. , 99 1329 1334.
Bourgeois D. Ursby T. Wulff M. Pradervand C. Le Grand A. Schildkamp W. Laboure S. Srajer V. Teng T. Y. Roth M. Moffat K. 1996Feasibility and realization of single-pulse Laue diffraction on macromolecular crystals at ESRF. , 3 65 74.
Bratos S. Mirloup F. Vuilleumier R. Wulff M. Plech A. 2004X-ray "filming" of atomic motions in chemical reactions. , 304 245 251.
Cammarata M. Levantino M. Schotte F. Anfinrud P. A. Ewald F. Choi J. Cupane A. Wulff M. Ihee H. 2008Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering. , 5 881 886.
Cammarata M. Lorenc M. Kim T. K. Lee J. H. Kong Q. Y. Pontecorvo E. Lo Russo. M. Schiro G. Cupane A. Wulff M. Ihee H. 2006Impulsive solvent heating probed by picosecond x-ray diffraction. , 124, 124504.
Cavalieri A. L. Fritz D. M. Lee S. H. Bucksbaum P. H. Reis D. A. Rudati J. Mills D. M. Fuoss P. H. Stephenson G. B. Kao C. C. Siddons D. P. Lowney D. P. Mac Phee. A. G. Weinstein D. Falcone R. W. Pahl R. Als-Nielsen J. Blome C. Dusterer S. Ischebeck R. Schlarb H. Schulte-Schrepping H. Tschentscher T. Schneider J. Hignette O. Sette F. Sokolowski-Tinten K. Chapman H. N. Lee R. W. Hansen T. N. Synnergren O. Larsson J. Techert S. Sheppard J. Wark J. S. Bergh M. Caleman C. Huldt G. van der Spoel D. Timneanu N. Hajdu J. Akre R. A. Bong E. Emma P. Krejcik P. Arthur J. Brennan S. Gaffney K. J. Lindenberg A. M. Luening K. Hastings J. B. 2005Clocking femtosecond x rays. , 94.
Cavalleri A. Wall S. Simpson C. Statz E. Ward D. W. Nelson K. A. Rini M. Schoenlein R. W. 2006Tracking the motion of charges in a terahertz light field by femtosecond X-ray diffraction. , 442 664 666.
Chen E. Wood M. J. Fink A. L. Kliger D. S. 1998Time-Resolved Circular Dichroism Studies of Protein Folding Intermediates of Cytochrome c. , 37 5589 5598.
Chen L. X. Jager W. J. H. Jennings G. Gosztola D. J. Munkholm A. Hessler J. P. 2001Capturing a Photoexcited Molecular Structure Through Time-Domain X-ray Absorption Fine Structure. , 292 262 264.
Christensen M. Haldrup K. Bechgaard K. Feidenhans’l R. Kong Q. Y. Cammarata M. Lo Russo. M. Wulff M. Harrit N. Nielsen M. M. 2009Time-Resolved X-ray Scattering of an Electronically Excited State in Solution. Structure of the (3)A(2u) State of Tetrakis-mu-pyrophosphitodiplatinate(II). , 131 502 508.
Collet E. Lemee-Cailleau M. H. Buron Le Cointe. M. Cailleau H. Wulff M. Luty T. Koshihara S. Y. Meyer M. Toupet L. Rabiller P. Techert S. 2003Laser-induced ferroelectric structural order in an organic charge-transfer crystal. , 300 612 615.
Coppens P. 2003What can time-resolved diffraction tell us about transient species? excited-state structure determination at atomic resolution. , 1317 1320.
Coppens P. Vorontsov I. I. Graber T. Kovalevsky A. Y. Chen Y. S. Wu G. Gembicky M. Novozhilova I. V. 2004Geometry changes of a Cu(I) phenanthroline complex on photoexcitation in a confining medium by time-resolved x-ray diffraction. , 126 5980 5981.
Davidsson J. Poulsen J. Cammarata M. Georgiou P. Wouts R. Katona G. Jacobson F. Plech A. Wulff M. Nyman G. Neutze R. 2005Structural determination of a transient isomer of CH2I2 by picosecond x-ray diffraction. , 94, 245503.
Fritz D. M. Reis D. A. Adams B. Akre R. A. Arthur J. Blome C. Bucksbaum P. H. Cavalieri A. L. Engemann S. Fahy S. Falcone R. W. Fuoss P. H. Gaffney K. J. George M. J. Hajdu J. Hertlein M. P. Hillyard P. B. Hoegen M. H. V. Kammler M. Kaspar J. Kienberger R. Krejcik P. Lee S. H. Lindenberg A. M. Mc Farland B. Meyer D. Montagne T. Murray E. D. Nelson A. J. Nicoul M. Pahl R. Rudati J. Schlarb H. Siddons D. P. Sokolowski-Tinten K. Tschentscher T. von der Linde. D. Hastings J. B. 2007Ultrafast bond softening in bismuth: Mapping a solid’s interatomic potential with X-rays. , 315 633 636.
Gaffney K. J. Lindenberg A. M. Larsson J. Sokolowski-Tinten K. Blome C. Synnergren O. Sheppard J. Caleman C. Mac Phee. A. G. Weinstein D. Lowney D. P. Allison T. Matthews T. Falcone R. W. Cavalieri A. L. Fritz D. M. Lee S. H. Bucksbaum P. H. Reis D. A. Rudati J. Macrander A. T. Fuoss P. H. Kao C. C. Siddons D. P. Pahl R. Moffat K. Als-Nielsen J. Duesterer S. Ischebeck R. Schlarb H. Schulte-Schrepping H. Schneider J. von der Linde. D. Hignette O. Sette F. Chapman H. N. Lee R. W. Hansen T. N. Wark J. S. Bergh M. Huldt G. van der Spoel D. Timneanu N. Hajdu J. Akre R. A. Bong E. Krejcik P. Arthur J. Brennan S. Luening K. Hastings J. B. 2005Observation of structural anisotropy and the onset of liquidlike motion during the nonthermal melting of InSb. , 95.
Gembicky M. Adachi S. Coppens P. 2007A kHz heat-load shutter for white-beam experiments at synchrotron sources. , 14 295 296.
Georgiou P. Vincent J. Andersson M. Wohri A. B. Gourdon P. Poulsen J. Davidsson J. Neutze R. 2006Picosecond calorimetry: Time-resolved x-ray diffraction studies of liquid CH2Cl2. , 124, 234507.
Grishaev A. Wu J. Trewhella J. Bax A. 2005Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data. , 127 16621 16628.
Haldrup T. Lemke H. T. Haldrup K. Nielsen T. N. Arms D. A. Walko D. A. Miceli A. Landahl E. C. Dufresne E. M. Nielsen M. M. 2009Picosecond time-resolved laser pump/X-ray probe experiments using a gated single-photon-counting area detector. , 16 387 390.
Ichiyanagi K. Sato T. Nozawa S. Kim K. H. Lee J. H. Choi J. Tomita A. Ichikawa H. Adachi S. Ihee H. Koshihara S. 2009100-picosecond time-resolved solution scattering utilizing wide-bandwidth X-ray beam from multilayer optics. , 16 391 394.
Ihee H. 2009Visualizing Solution-Phase Reaction Dynamics with Time-Resolved X-ray Liquidography. , 42 356 366.
Ihee H. Lorenc M. Kim T. K. Kong Q. Y. Cammarata M. Lee J. H. Bratos S. Wulff M. 2005aUltrafast x-ray diffraction of transient molecular structures in solution. , 309 1223 1227.
Ihee H. Rajagopal S. Srajer V. Pahl R. Anderson S. Schmidt M. Schotte F. Anfinrud P. A. Wulff M. Moffat K. 2005bVisualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. , 102 7145 7150.
Jones C. M. Henry E. R. Hu Y. C. Chan K. Luck S. D. A. A. B. Roder H. Hofrichter J. Eaton W. A. 1993Fast events in protein folding initiated by nanosecond laser photolysis. , 90 11860 11864.
Kainosho M. Torizawa T. Iwashita Y. Terauchi T. Ono A. M. P. P. G. 2006Optimal isotope labelling for NMR protein structure determinations. , 440 52 57.
Kim C. D. Pillet S. Wu G. Fullagar W. K. Coppens P. 2002Excited-state structure by time-resolved X-ray diffraction. , 58 133 137.
Kim K. J. Shvyd’ko Y. Reiche S. 2008A proposal for an X-ray free-electron laser oscillator with an energy-recovery linac. , 100, 244802.
Kim T. K. Lorenc M. Lee J. H. Russo M. Kim J. Cammarata M. Kong Q. Y. Noel S. Plech A. Wulff M. Ihee H. 2006Spatiotemporal reaction kinetics of an ultrafast photoreaction pathway visualized by time-resolved liquid x-ray diffraction. , 103 9410 9415.
Kong Q. Lee J. H. Plech A. Wulff M. Ihee H. Koch M. H. J. 2008Ultrafast X-ray Solution Scattering Reveals an Unknown Reaction Intermediate in the Photolysis of Ru3(CO)12. , 47 5550 5553.
Kong Q. Y. Wulff M. Lee J. H. Bratos S. Ihee H. 2007Photochemical reaction pathways of carbon tetrabromide in solution probed by picosecond X-ray diffraction. , 129 13584 13591.
Lee J. H. Kim J. Cammarata M. Kong Q. Kim K. H. Choi J. Kim T. K. Wulff M. Ihee H. 2008aTransient X-ray diffraction reveals global and major reaction pathways for the photolysis of iodoform in solution. , 47 1047 1050.
Lee J. H. Kim K. H. Kim T. K. Lee Y. Ihee H. 2006Analyzing solution-phase time-resolved x-ray diffraction data by isolated-solute models. , 125, 17450.
Lee J. H. Kim T. K. Kim J. Kong Q. Cammarata M. Lorenc M. Wulff M. Ihee H. 2008bCapturing transient structures in the elimination reaction of haloalkane in solution by transient X-ray diffraction. , 130 5834 5835.
Lee S. H. Cavalieri A. L. Fritz D. M. Swan M. C. Hegde R. S. Reason M. Goldman R. S. Reis D. A. 2005Generation and propagation of a picosecond acoustic pulse at a buried interface: Time-resolved X-ray diffraction measurements. , 95.
Lindenau B. Räbiger J. Polachowski S. Fremerey J. K. 2004X-ray pulse selector with 2 ns lock-in phase setting and stability. , 705 1019 1022.
Lindenberg A. M. Larsson J. Sokolowski-Tinten K. Gaffney K. J. Blome C. Synnergren O. Sheppard J. Caleman C. Mac Phee. A. G. Weinstein D. Lowney D. P. Allison T. K. Matthews T. Falcone R. W. Cavalieri A. L. Fritz D. M. Lee S. H. Bucksbaum P. H. Reis D. A. Rudati J. Fuoss P. H. Kao C. C. Siddons D. P. Pahl R. Als-Nielsen J. Duesterer S. Ischebeck R. Schlarb H. Schulte-Schrepping H. Tschentscher T. Schneider J. von der Linde. D. Hignette O. Sette F. Chapman H. N. Lee R. W. Hansen T. N. Techert S. Wark J. S. Bergh M. Huldt G. van der Spoel D. Timneanu N. Hajdu J. Akre R. A. Bong E. Krejcik P. Arthur J. Brennan S. Luening K. Hastings J. B. 2005Atomic-scale visualization of inertial dynamics. , 308 392 395.
Moffat K. 2001Time-resolved biochemical crystallography: a mechanistic perspective. , 101 1569 1581.
Mukamel S. Abramavicius D. Yang L. Zhuang W. Schweigert I. V. Voronine D. V. 2009Coherent multidimensional optical probes for electron correlations and exciton dynamics: From NMR to X-rays. , 42 553 562.
Nozawa S. Adachi S. Takahashi J. Tazaki R. Guerin L. Daimon M. Tomita A. Sato T. Chollet M. Collet E. Cailleau H. Yamamoto S. Tsuchiya K. Shioya T. Sasaki H. Mori T. Ichiyanagi K. Sawa H. Kawata H. Koshihara S. 2007Developing 100 ps-resolved X-ray structural analysis capabilities on beamline NW14A at the photon factory advanced ring. , 14 313 319.
Plech A. Wulff M. Bratos S. Mirloup F. Vuilleumier R. Schotte F. Anfinrud P. A. 2004Visualizing chemical reactions in solution by picosecond x-ray diffraction. , 92, 125505.
Saes M. Bressler C. Abela R. Grolimund D. Johnson S. L. Heimann P. A. Chergui M. 2003Observing Photochemical Transients by Ultrafast X-Ray Absorption Spectroscopy. , 90, 047403.
Sato T. Nozawa S. Ichiyanagi K. Tomita A. Chollet M. Ichikawa H. Fujii H. Adachi S. Koshihara S. 2009Capturing molecular structural dynamics by 100 ps time-resolved X-ray absorption spectroscopy. , 16 110 115.
Schotte F. Lim M. Jackson T. A. Smirnov A. V. Soman J. Olson J. S. Phillips G. N. J. Wulff M. Anfinrud P. 2003Watching a protein as it functions with 150ps time-resolved X-ray crystallography. , 300 1944 1947.
Schwieters C. D. Kuszewski J. J. Tjandra N. Clore G. M. 2003The Xplor-NIH NMR molecular structure determination package. , 160 65 73.
Srajer V. Teng T. Y. Ursby T. Pradervand C. Ren Z. Adachi S. Schildkamp W. Bourgeois D. Wulff M. Moffat K. 1996Photolysis of the carbon monoxide complex of myoglobin: Nanosecond time-resolved crystallography. , 274 1726 1729.
Techert S. Schotte F. Wulff M. 2001Picosecond X-Ray Diffraction Probed Transeint Structural Changes in Organic Solids. , 86 2030 2033.
Tomita A. Sato T. Ichiyanagi K. Nozawa S. Ichikawa H. Chollet M. Kawai F. Park S. Y. Tsuduki T. Yamato T. Koshihara S. Adachi S. 2009Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin. , 106 2612 2616.
Vincent J. Andersson M. Eklund M. Wohri A. B. Odelius M. Malmerberg E. Kong Q. Y. Wulff M. Neutze R. Davidsson J. 2009Solvent dependent structural perturbations of chemical reaction intermediates visualized by time-resolved x-ray diffraction. , 130,-.
Wall M. Tarnovsky A. N. Pascher T. Sundstrom V. Akesson E. 2003Photodissociation Dynamics of Iodoform in Solution. , 107 211 217.
Wulff M. Bratos S. Plech A. Vuilleumier R. Mirloup F. Lorenc M. Kong Q. Ihee H. 2006Recombination of photodissociated iodine: A time-resolved x-ray-diffraction study. , 124, 034501.
Wulff M. Schotte F. Naylor G. Bourgeois D. Moffat K. Mourou G. 1997Time-resolved structures of macromolecules at the ESRF: Single-pulse Laue diffraction, stroboscopic data collection and femtosecond flash photolysis. , A 398 69 84.
Zheng X. M. Phillips D. L. 2000Solvation effects on the iodoform ultraviolet direct photodissociation reaction. opening the photoisomerization channel. , 324 175 182.