Diffracted X-ray Tracking Method Research Articles

Real-time observation of capsaicin-induced domain dynamics of TRPV1 using the diffracted X-ray tracking method

Real-time observation of capsaicin-induced domain dynamics of TRPV1 using the diffracted X-ray tracking method

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer’s disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Analysis of the Structural Dynamics of Proteins in the Ligand-Unbound and -Bound States by Diffracted X-ray Tracking.

Although many protein structures have been determined at atomic resolution, the majority of them are static and represent only the most stable or averaged structures in solution. When a protein binds to its ligand, it usually undergoes fluctuation and changes its conformation. One attractive method for obtaining an accurate view of proteins in solution, which is required for applications such as the rational design of proteins and structure-based drug design, is diffracted X-ray tracking (DXT). DXT can detect the protein structural dynamics on a timeline via gold nanocrystals attached to the protein. Here, the structure dynamics of single-chain Fv antibodies, helix bundle-forming de novo designed proteins, and DNA-binding proteins in both ligand-unbound and ligand-bound states were analyzed using the DXT method. The resultant mean square angular displacements (MSD) curves in both the tilting and twisting directions clearly demonstrated that structural fluctuations were suppressed upon ligand binding, and the binding energies determined using the angular diffusion coefficients from the MSD agreed well with the binding thermodynamics determined using isothermal titration calorimetry. In addition, the size of gold nanocrystals is discussed, which is one of the technical concerns of DXT.

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.

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.

Dynamical Analysis of Peptide Exchange Mechanism of HLA-DM by Diffracted X-ray Tracking Method

Dynamical Analysis of Peptide Exchange Mechanism of HLA-DM by Diffracted X-ray Tracking Method

Diffracted X-ray tracking method for recording single-molecule protein motions

BackgroundProteins change their conformation depending on function. Although a vast number of static pictures of proteins have been accumulated, information regarding their dynamics in function is limited. Diffracted X-ray tracking (DXT) is a good candidate to obtain the missing data. Scope of reviewA gold nanocrystal was attached to the target protein as a probe and the motion of the X-ray diffraction spots from the crystal corresponded to the motion of the target. Although it has advantages of high temporal (sub-millisecond) and spatial (approximately 0.1°) resolutions, it is not extensively utilized. This review focused on its effective application from a user's perspective. We also present an example with the KcsA channel and the status of recent developments to show the future possibilities of the method. Major conclusionsDXT is a powerful method to investigate intramolecular structural changes. For instance, in the KcsA channel, the method revealed a wave of conformational changes transmitted from the gate region to the end of the molecule. The method is continuously being developed, and users can choose an appropriate measurement system depending on the condition of their sample. General significanceRevealing the protein structural changes with respect to function is an important frontier. The most distinctive feature of the DXT method is that both high temporal and spatial resolutions are achievable, and it is possible to track the motions of multiple molecules at the same time. This feature is an advantage for screening molecules associated with the target proteins (e.g., ligands and medicines).

DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking

Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method

The TRPV1 is a nonselective cation channel that responds to various signals, including high temperature with threshold at 43 °C, acidic conditions, as well as chemical compounds such as capsaicin and allylisothiocyanate. Because TRPV1 is a membrane protein with large molecular mass, crystallography technique has not been succeeded for a long time. However recent progress in the cryoelectron microscopy enabled depiction of the TRPV1 structure in atomic resolution. Cryo-EM and the single particle reconstruction technique showed structures of apo- and ligand binding forms of TRPV1 with various agonists, antagonists and toxins. In spite of accumulation of structural data, gating mechanisms of TRPV1 is not clearly understood yet. Information about state-to-state transition is missing. To understand the dynamics of TRPV1 in channel function, we adopted the Diffracted X-ray Tracking (DXT) technique to this protein. In DXT, individual protein was labeled with gold nanocrystals, and the motion of X-ray diffraction spots from the crystal were investigated as intramolecular movement of TRPV1 in real time. We introduced “Met tag” for labeling nanocrystal and “His tag” for substrate absorption to TRPV1. It was expressed in HEK293 cells, purified, and immobilized on the Ni-NTA coated polyimide substrates. Intramolecular motion was recorded by tracking the movement of diffraction spots from the gold nanocrystals. Molecular dynamics of TRPV1 against capsaicin, pH change, and temperature activation were investigated.

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

Photoresist Micro-Chamber for the Diffracted X-ray Tracking Method Recording Single-Molecule Conformational Changes

The diffracted X-ray tracking method has been established for capturing conformational changes of single KcsA potassium channels, which records position of X-ray diffraction spot from a gold nanocrystal attached to the channel. The conventional observation chamber setup could trace a diffraction spot; however relatively high background noise resulting from X-ray scattering degraded a spatial resolution. This paper report a novel low-noise microfabricated observation chamber consisting of SiN membrane window supported by Si and embedded microchannel in negative photoresist. The measured noise exhibited lower level compared to the conventional chamber, which almost reached the detection limit of the employed imaging system.

Microsecond X-Ray Dynamics Observation of Nano Supersaturated Protein's Network

Today, comprehension of supersaturated phenomena in protein solution is one of the most important issue. For example, elucidation of amyloid fibril formation process which is known as a main causative agent causing Alzheimer disease[1] or bio-mineralization process of CaCO[2]. In a recent study, molecular dynamics simulation (MD) for crystallization process of CaCO3 was conducted by A.F. Wallace et al[3]. They simulated that the supersaturated solution has a local metastable state as a microscopic ion cluster (we called nano supersaturated network). On the other hand, until now, an experimental detection system for the nano supersaturated network has not been established so far.In our study, we utilized Diffracted X-ray Tracking (DXT) as a high time resolution (25µs) and high positional accuracy (-pm) detection system for the time resolved dynamical interactions between a single gold nanocrystal(-80nm) and localized prenucleation clusters as a nano supersaturated network at SPring-8(BL40XU). For the target samples, we choose two samples that are the sodium acetate trihydrate and lysozyme supersaturated solution as an inorganic and protein solution system, respectively. From DXT result, we succeeded in observing the localized high and low speed motions of gold nanocrystals interacted with nano supersaturated protein's networks. At this presentation, I explain the DXT method and obtained physical parameters of nano supersaturated networks in inorganic and protein supersaturated solution system from DXT analysis. In finally, I present a comprehensive model of nano supersaturated networks.

3P030 高時間分解能で蛋白質の分子揺らぎと構造変化を計測するためのX線1分子動態計測法の開発(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

3P030 高時間分解能で蛋白質の分子揺らぎと構造変化を計測するためのX線1分子動態計測法の開発(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

Dynamical Peptide Motions in MHC Groove is Crucial for Recognition and Activation of Certain T Cells

We detected dynamic motions of peptides in MHC II with single-molecule technology using X-rays. T cells see peptides in the context of MHC molecules. This recognition should be specific to each antigens to prevent anomalous self-attack by the activated T cells. However, cross-reactivity of T cell is also well known phenomena. Also, some T cells see peptides/MHC in peculiar forms. As a typical of such peptide/MHC, we studied type B form of I-Ak with HEL peptides, as well as I-Ag7 with newly found diabetogenic peptides for molecular flexibility. in order to elucidate the mechanism of the recognition, we used diffracted X-ray tracking method (DXT) that monitors real-time movements of individual proteins in solution at the single-molecular level. We found that peptides have its individual Brownian motions that are different from MHC protein themself, and the extent of motion diminishes by time in order of days, which may mimic the DM function. We also found that the rotational motions of peptides correlate with the type B T cell activation or with diabetogenic I-Ag7 activation. The rotational motion of peptides may create transient conformation of peptide/MHC complex that recognized by a population of T cells.

Laser-Triggered Single Molecular Gating Motions of the KcsA Potassium Channels Recorded in a Sub-Millisecond Time Resolution

We previously revealed that the KcsA potassium channel exhibited global twisting motions upon gating (Shimizu, H., et al Cell 132, 67-78, 2008). The method we used was the diffracted X-ray tracking (DXT), in which the conformational changes were recorded under equilibrium conditions at video rate. To trace the whole picture of the gating motions, further development of the method is required. We recently have succeeded in developing a laser-triggered measurement system, while the X-ray spectrum was optimized for fast recordings with a high-speed camera. The channel was fixed on a glass plate at the extracellular loop and a gold nanocrystal was attached to the C-terminal end of the channel molecule as a probe. The sample was irradiated with an optimized white-beam from a synchrotron, and the motions of the Laue diffraction spots from the nanocrystal were tracked on a CMOS detector at the speed of 5000 frames/s. During the measurements the pH of the solution was changed from neutral to acidic by laser-photolysis of the caged proton, which triggers the gating motions of the pH-gated KcsA channel. Trajectories of the diffraction spots on the detector plane were translated as movements of the channel in the real space. This modified DXT method now enables us to detect the initial opening processes of the conformational changes in the time resolution comparable to that of the single channel current recordings.

Direct Observation of Torsional Motion of Group II Chaperonin

Diffracted X-ray Tracking (DXT) has been considered as a powerful technique in biological science for detecting subtle (pico meter scale) dynamic motion of the target protein at single molecular level. This method was applied for various proteins, such as bacteriorhodopsin [1], antibody [2] and KcsA channel [3]. In DXT, the dynamics of a single protein can be monitored through trajectory of the Laue spot from the nanocrystal which was labeled on the objective protein immobilized on the substrate surface.In this study, DXT method was applied to the group II chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an ATP dependent manner [4]. A mutant group II chaperonin from Thermococcus strain KS-1 with a Cys residue at the tip of the helical protrusion, was immobilized on the gold coated substrate surface and was labeled with a gold nanocrystal through gold-thiol bond.We monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the rotational motion of the nanocrystal, which corresponded to the torsional motion of the chaperonin, in the presence of ATP condition was 10 times larger than that in the absence of ATP condition. And UV-light triggered DXT study using caged ATP revealed that the chaperonin twisted counterclockwisely (from the top to the bottom view of chaperonin) when ATP binded to the chaperonin and the angular velocity from open to closed state of chaperonin chamber was 10 % faster than that from closed to open state.[1] Y. Okumura et al., Phys. Rev. E, 70:021917 (2004)[2] T. Sagawa et al., Biochem. Biophys. Res. Commun. 335:770 (2007)[3] H. Shimizu et al., Cell 132:67 (2008)[4] T. Kanzaki et al., J. Biol. Chem. 283: 34773 (2008)

The Intra Dynamics of Group II Chaperonin Detected by Diffracted X-Ray Tracking Method

Besides the static structural information of a protein, it is crucial for revealing mechanism of protein's function that to pursue the intra-dynamics information of the objective protein in millisecond and atomic scale.We had proposed the Diffracted X-ray Tracking (DXT) for detecting subtle intra-movement of the target protein, and applied this method for some proteins, such as bactriorhodopsin [1], antibody [2] and KcsA channel [3]. In DXT, the dynamics of a single protein can be monitored through trajectory of the Laue spots from the nano crystal which was labeled on the objective proteins immobilized on the substrate surface.In this study, we applied the DXT method for monitoring ATP driven conformational change of archaeal group II chaperonin, known as the protein machinery that interacts with misfolded proteins, confine them in its cavity by closure of the built-in lid, and assists them to re-fold correctly in the cavity [4].Optimizing the experimental condition of DXT method, such as immobilization of proteins and prepation of gold nanocrystals, the dynamics of open and closure of the chaperonin's built-in lid will be discussed.[1] Y. Okumura et al., Phys. Rev. E, 70:021917-1-7 (2004)[2] T. Sagawa et al., Biochem. Biophy. Res. Commun. 335:770-775 (2007)[3] H. Shimizu et al., Cell 132:67-78 (2008)[4] T. Kanzaki et al., J. Biol. Chem. 283:34773-34784