Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

Sekiguchi, Hiroshi; Suzuki, Yasuhito; Nishino, Yuri; Kobayashi, Suzuko; Shimoyama, Yoshiko; Cai, Wei; Nagata, Kenji; Okada, Masato; Ichiyanagi, Kouhei; Ohta, Nobuhiro; Yagi, Naoto; Miyazawa, Atsuo; Kubo, Tai; Sasaki, Y.

doi:10.1038/srep06384

Cited by 40 publications

(56 citation statements)

References 35 publications

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“…In this study, we developed a novel method for dynamic observation of local supersaturated solutions at microsecond time scales using an X-ray single molecule methodology, also known as diffracted X-ray tracking (DXT) 15 16 17 . In particular, DXT can be used to detect high accuracy rotational angular observations in solution systems.…”

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confidence: 99%

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Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks

Matsushita

Sekiguchi

Ichiyanagi

et al. 2015

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Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 μs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

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confidence: 99%

“…We utilized DXT as a high time resolution (μs) and high positional accuracy (pm) 17 X-ray single molecule observation method to investigate the properties of supersaturated solutions ( Fig. 1a ).…”

mentioning

confidence: 99%

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks

Matsushita

Sekiguchi

Ichiyanagi

et al. 2015

Sci Rep

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“…However, previous attempts of AChBP expression in E . coli resulted in accumulation of these proteins in inclusion bodies [ 38 ]. This necessitated the use of expensive and time-consuming re-solubilization techniques to recover the expressed protein.…”

Section: Discussionmentioning

confidence: 99%

“…Previous attempts to express the AChBPs in E . coli produced only insoluble AChBP, requiring expensive and time-consuming stabilization and refolding steps to obtain functional protein [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

et al. 2016

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Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

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“…This diffracted X-ray tracking 5 – 7 (DXT) method can trace rotational motions within single protein molecules using X-rays with a wide energy bandwidth. Among various applications, DXT has been utilised to monitor the dynamic twisting motions of membrane proteins 8 , 9 chaperonin proteins 10 , 11 or other globular macromolecules 12 – 14 . This technique is precise to a sub-mrad angular scale.…”

Section: Introductionmentioning

confidence: 99%

Diffracted X-ray Blinking Tracks Single Protein Motions

Sekiguchi

Kawamura

Ikezaki

et al. 2018

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Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.

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Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

Cited by 40 publications

References 35 publications

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks

Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

Diffracted X-ray Blinking Tracks Single Protein Motions

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