Simultaneous Recording of Remote Domain Dynamics in Membrane Proteins Using the Double-Labeled DXB/DXT Technique.

Protein dynamics play important roles in biological functions, which accompany allosteric structure changes. Diffracted X-ray blinking (DXB) uses monochromatic X-rays and nanocrystal probes. The intramolecular motion of target proteins is analyzed from the intensity changes in detector signals at the diffraction rings. In contrast, diffracted X-ray tracking (DXT) elucidates molecular dynamics by analyzing the trajectories of Laue spots. In this study, we have developed a dual-labeling technique for DXB and DXT, allowing the simultaneous observation of motions at different domains in proteins. We identified zinc oxide (ZnO) crystals as promising candidates for the second labeling probes due to their excellent diffraction patterns, high chemical stability, and favorable binding properties with proteins. The diffraction spots from the ZnO crystals are sufficiently separated from those of gold, enabling independent motion analysis at different domains. Dual-labeling DXB was employed for the motion analysis of the 5-HT2A receptor in living cells. Simultaneous motion recording of the N-terminus and the second extracellular loop demonstrated ligand-induced motion suppression at both domains. The dual-labeling DXT technique demonstrated a capsaicin-induced peak shift in the two-dimensional motion maps at the N-terminus of the TRPV1 protein, but the peak shift was not obvious in the C-terminus. The capsaicin-induced motion modulation was recovered by the addition of the competitive inhibitor AMG9810.

Real-Time Observation of Capsaicin-Induced Intracellular Domain Dynamics of TRPV1 Using the Diffracted X-ray Tracking Method.

The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 µs/frame and 12.5 ms/frame. The data from the 100 µs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 µs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

Dynamic observations of various oligomers in amyloid ß isoforms using laboratory diffracted X-ray blinking.

Acceleration of societal ageing has increased the global incidence of geriatric diseases such as Alzheimer's disease (AD), and the demands for proper diagnosis and monitoring of those diseases are also increasing daily. We utilized diffracted X-ray blinking (DXB) for amyloid ß (Aß) isoforms, which are thought to be closely related to AD, to discriminate among the dynamics of individual particles in early and long-term oligomerisation and aggregation inhibiting environments. Among the various Aß isoforms, the dynamics of Aß (1-42), which is known to be the most toxic form, were the slowest (the dynamics were lower by 78% com-pared with short-term incubation), and the dynamics were restored (the dynamics increased by 105% compared with normal aggregation) in an environment that suppressed oligomerisation of Aß (1-42). It has been confirmed that the use of DXB allows measurements of dynamics related to the functional states of the target molecules.

Diffracted X-ray Tracking Method for Measuring Intramolecular Dynamics of Membrane Proteins.

Membrane proteins change their conformations in response to chemical and physical stimuli and transmit extracellular signals inside cells. Several approaches have been developed for solving the structures of proteins. However, few techniques can monitor real-time protein dynamics. The diffracted X-ray tracking method (DXT) is an X-ray-based single-molecule technique that monitors the internal motion of biomolecules in an aqueous solution. DXT analyzes trajectories of Laue spots generated from the attached gold nanocrystals with a two-dimensional axis by tilting (θ) and twisting (χ). Furthermore, high-intensity X-rays from synchrotron radiation facilities enable measurements with microsecond-timescale and picometer-spatial-scale intramolecular information. The technique has been applied to various membrane proteins due to its superior spatiotemporal resolution. In this review, we introduce basic principles of DXT, reviewing its recent and extended applications to membrane proteins and living cells, respectively.