X-ray-based living-cell motion analysis of individual serotonin receptors.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells. In this study, we used Diffracted X-ray Tracking (DXT) and Diffracted X-ray Blinking (DXB) techniques for analysis. These methods are very precise single-molecular analytical techniques that elucidate protein dynamics by analyzing the diffraction spots from the gold nanocrystals labeled on the protein surface. DXT tracks diffraction spot movements, whereas DXB analyzes continuation of signals by calculating the autocorrelation function of each pixel from the recorded data. Serotonin receptor subtype 2A (5-HT2A receptors) were transiently expressed on HEK 293 cells, and the gold nanocrystals were attached to the N-terminally introduced FLAG-tag via anti-FLAG antibodies. Fast- and mid-range motions were recorded by DXT with 100µs and 1.25 ms/frame rate, respectively. Slow-range motion was obtained using the DXB method with 100 ms/frame rate. An agonist interestingly suppressed the fluctuations of 5-HT2A receptors at the microsecond-ranged fast measurement. On the contrary, the motion was enhanced by the agonist in the hundred-millisecond-ranged slow time scale. These dual-natured data may suggest that we succeeded in extracting different modes of receptor's motion on live cells; microsecond ranged fluctuation on the cell membrane, and millisecond-ranged dynamic movement comprising interactions with intracellular signaling molecules.

Photoinduced anisotropic distortion as the electron trapping site of tungsten trioxide by ultrafast W L1-edge X-ray absorption spectroscopy with full potential multiple scattering calculations.

Understanding the excited state of photocatalysts is significant to improve their activity for water splitting reaction. X-ray absorption fine structure (XAFS) spectroscopy in X-ray free electron lasers (XFEL) is a powerful method to address dynamic changes in electronic states and structures of photocatalysts in the excited state in ultrafast short time scales. The ultrafast atomic-scale local structural change in photoexcited WO3 was observed by W L1 edge XAFS spectroscopy using an XFEL. An anisotropic local distortion around the W atom could reproduce well the spectral features at a delay time of 100 ps after photoexcitation based on full potential multiple scattering calculations. The distortion involved the movement of W to shrink the shortest W-O bonds and elongate the longest one. The movement of the W atom could be explained by the filling of the dxy and dzx orbitals, which were originally located at the bottom of the conduction band with photoexcited electrons.

Selective Reduction Mechanism of Graphene Oxide Driven by the Photon Mode versus the Thermal Mode.

A two-dimensional nanocarbon, graphene, has attracted substantial interest due to its excellent properties. The reduction of graphene oxide (GO) has been investigated for the mass production of graphene used in practical applications. Different reduction processes produce different properties in graphene, affecting the performance of the final materials or devices. Therefore, an understanding of the mechanisms of GO reduction is important for controlling the properties of functional two-dimensional systems. Here, we determined the average structure of reduced GO prepared via heating and photoexcitation and clearly distinguished their reduction mechanisms using ultrafast time-resolved electron diffraction, time-resolved infrared vibrational spectroscopy, and time-dependent density functional theory calculations. The oxygen atoms of epoxy groups are selectively removed from the basal plane of GO by photoexcitation (photon mode), in stark contrast to the behavior observed for the thermal reduction of hydroxyl and epoxy groups (thermal mode). The difference originates from the selective excitation of epoxy bonds via an electronic transition due to their antibonding character. This work will enable the preparation of the optimum GO for the intended applications and expands the application scope of two-dimensional systems.

X-ray observations of single bio-supramolecular photochirogenesis.

The binding and photochirogenic behaviour of 2-anthracenecarboxylate (AC) with human serum albumin (HSA) have hitherto been investigated and comprehended as time-averaged statistical events by spectroscopic examinations and product analyses. In this study, we employed a diffracted X-ray tracking (DXT) technique to visualize the single-molecular dynamics of free and AC-loaded HSA (AC:HSA = 0, 1, 5 and 10), as well as the AC-HSA complex under photoirradiation, all of which were tethered to gold nanocrystals and hence traceable in real time by DXT. This enabled us to draw a more dynamic picture of the bio-supramolecular photochirogenesis at a single-molecule resolution, detailing the softening and flexibility enhancement of HSA upon binding of ACs to its inter-subdomain IIA-IIB site and the dynamic extrusion of AC dimers produced upon photoirradiation.

Capturing local structure modulations of photoexcited BiVO4 by ultrafast transient XAFS.

Ultrafast excitation of photocatalytically active BiVO4 was characterized by femto- and picosecond transient X-ray absorption fine structure spectroscopy. An initial photoexcited state (≪500 fs) changed to a metastable state accompanied by a structural change with a time constant of ∼14 ps. The structural change might stabilize holes on oxygen atoms since the interaction between Bi and O increases.

Dynamics of Photoelectrons and Structural Changes of Tungsten Trioxide Observed by Femtosecond Transient XAFS.

The dynamics of the local electronic and geometric structures of WO3 following photoexcitation were studied by femtosecond time-resolved X-ray absorption fine structure (XAFS) spectroscopy using an X-ray free electron laser (XFEL). We found that the electronic state was the first to change followed by the local structure, which was affected within 200 ps of photoexcitation.

The RATIO method for time-resolved Laue crystallography.

A RATIO method for analysis of intensity changes in time-resolved pump-probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam.

Capturing molecular structural dynamics by 100 ps time-resolved X-ray absorption spectroscopy.

An experimental set-up for time-resolved X-ray absorption spectroscopy with 100 ps time resolution at beamline NW14A at the Photon Factory Advanced Ring is presented. The X-ray positional active feedback to crystals in a monochromator combined with a figure-of-merit scan of the laser beam position has been utilized as an essential tool to stabilize the spatial overlap of the X-ray and laser beams at the sample position. As a typical example, a time-resolved XAFS measurement of a photo-induced spin crossover reaction of the tris(1,10-phenanthrorine)iron(II) complex in water is presented.

Developing 100 ps-resolved X-ray structural analysis capabilities on beamline NW14A at the Photon Factory Advanced Ring.

NW14A is a newly constructed undulator beamline for 100 ps time-resolved X-ray experiments at the Photon Factory Advanced Ring. This beamline was designed to conduct a wide variety of time-resolved X-ray measurements, such as time-resolved diffraction, scattering and X-ray absorption fine structure. Its versatility is allowed by various instruments, including two undulators, three diffractometers, two pulse laser systems and an X-ray chopper. The potential for the detection of structural changes on the 100 ps time scale at NW14A is demonstrated by two examples of photo-induced structural changes in an organic crystal and photodissociation in solution.