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Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 9910

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Prof. Dr. Kazuhiro Mio

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Guest Editor

National Institute of Advanced Industrial Science and Technology (AIST), 6-2-3 Kashiwanoha, Kashiwa 277-0882, Japan
Interests: structural biology; ion channel; electronic microscopy; single particle reconstruction; diffracted X-ray tracking; diffracted X-ray blinking

Prof. Dr. Yuji C. Sasaki

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Guest Editor

Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Chiba 277-8561, Japan
Interests: single molecule observations; intramolecular motion analysis; diffracted X-ray tracking; diffracted X-ray blinking; bio-nanotechnology

Special Issue Information

Dear Colleagues,

Recent technological advances, especially improvements in light sources, detector systems, and nanobiotechnology around sample preparations have revolutionized dynamic biomolecular analysis by capturing smaller signals at higher frame rates. Additionally, big data-driven science and technology have also improved dramatically. Researchers are improving the accuracy of data by actively utilizing machine learning and building a prediction system. X-rays are widely used for structural analysis due to their high penetrating power and crystal diffraction. Against this background, high-speed time-resolved analysis methods such as diffracted X-ray blinking (DXB), diffracted X-ray tracking (DXT), and X-ray free-electron laser (XFEL) are becoming a new generation. This Special Issue focuses on the direct measuring of the dynamic motion of biomolecules using quantum probes such as X-rays, electron beams and neutrons, and explains the functional mechanisms of target molecules on a real-time axis. Researchers in the biological sciences, organic and inorganic chemistry, and computational science are welcome to discuss topics that align with this Special Issue. Original papers, as well as review papers presenting future ideas, are welcome. We hope that all contributors and readers will be inspired by the bird's-eye view of various research themes.

Prof. Dr. Kazuhiro Mio
Prof. Dr. Yuji C. Sasaki
Guest Editors

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Keywords

single molecule measurement
molecular dynamics
intramolecular motion
motion tracking
structure and function
quantum probe
X-ray
electron beam
neutron

Published Papers (10 papers)

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Research

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13 pages, 2958 KiB

Open AccessArticle

Visualization of the Dynamics of Photoinduced Crawling Motion of 4-(Methylamino)Azobenzene Crystals via Diffracted X-ray Tracking

by Koichiro Saito, Kouhei Ichiyanagi, Ryo Fukaya, Rie Haruki, Shunsuke Nozawa, Daisuke Sasaki, Tatsuya Arai, Yuji C. Sasaki, Keegan McGehee, Makoto Saikawa, Minghao Gao, Zhichao Wei, Dennis Kwaria and Yasuo Norikane

Int. J. Mol. Sci. 2023, 24(24), 17462; https://doi.org/10.3390/ijms242417462 - 14 Dec 2023

Viewed by 662

Abstract

The photoinduced crawling motion of crystals is a continuous motion that azobenzene molecular crystals exhibit under light irradiation. Such motion enables object manipulation at the microscale with a simple setup of fixed LED light sources. Transportation of nano-/micromaterials using photoinduced crawling motion has recently been reported. However, the details of the motion mechanism have not been revealed so far. Herein, we report visualization of the dynamics of fine particles in 4-(methylamino)azobenzene (4-MAAB) crystals under light irradiation via diffracted X-ray tracking (DXT). Continuously repeated melting and recrystallization of 4-MAAB crystals under light irradiation results in the flow of liquid 4-MAAB. Zinc oxide (ZnO) particles were introduced inside the 4-MAAB crystals to detect diffracted X-rays. The ZnO particles rotate with the flow of liquid 4-MAAB. By using white X-rays with a wide energy width, the rotation of each zinc oxide nanoparticle was detected as the movement of a bright spot in the X-ray diffraction pattern. It was clearly shown that the ZnO particles rotated increasingly as the irradiation light intensity increased. Furthermore, we also found anisotropy in the rotational direction of ZnO particles that occurred during the crawling motion of 4-MAAB crystals. It has become clear that the flow perpendicular to the supporting film of 4-MAAB crystals is enhanced inside the crystal during the crawling motion. DXT provides a unique means to elucidate the mechanism of photoinduced crawling motion of crystals. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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17 pages, 3713 KiB

Open AccessArticle

Single-Molecule X-ray Scattering Used to Visualize the Conformation Distribution of Biological Molecules via Single-Object Scattering Sampling

by Seonggon Lee, Hosung Ki, Sang Jin Lee and Hyotcherl Ihee

Int. J. Mol. Sci. 2023, 24(24), 17135; https://doi.org/10.3390/ijms242417135 - 05 Dec 2023

Viewed by 781

Abstract

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as “Bio-SOSS”, leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA–protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 Å, typically within 1 Å under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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12 pages, 1130 KiB

Open AccessArticle

Ubiquitination of Major Histocompatibility Complex II Changes Its Immunological Recognition Structure

by Yuko Kozono, Masahiro Kuramochi, Yuji C. Sasaki and Haruo Kozono

Int. J. Mol. Sci. 2023, 24(23), 17083; https://doi.org/10.3390/ijms242317083 - 03 Dec 2023

Viewed by 730

Abstract

Ubiquitination is a process that dictates the lifespan of major histocompatibility complex class II (MHC II)/peptide complexes on antigen-presenting cells. This process is tightly controlled by the levels of ubiquitin ligases, and disruptions in the turnover of MHC II can lead to the improper development of CD4+ T cells within the thymus and hinder the formation of regulatory T cells in the peripheral tissue. To investigate the underlying mechanisms, we utilized dendritic cells lacking the Membrane-associated RING-CH (MARCH) I ubiquitin ligase. We discovered that the overexpression of MARCH I decreases the interaction with LAG-3. Moreover, the MHC II molecules tethered with ubiquitin also showed diminished binding to LAG-3. We employed Diffracted X-ray Blinking (DXB), a technique used for single-molecule X-ray imaging, to observe the protein movements on live cells in real time. Our observations indicated that the normal MHC II molecules moved more rapidly across the cell surface compared to those on the MARCH I-deficient dendritic cells or MHC II KR mutants, which is likely a result of ubiquitination. These findings suggest that the signaling from ubiquitinated MHC II to the T cell receptor differs from the non-ubiquitinated forms. It appears that ubiquitinated MHC II might not be quickly internalized, but rather presents antigens to the T cells, leading to a range of significant immunological responses. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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12 pages, 2712 KiB

Open AccessArticle

Time-Resolved X-ray Observation of Intracellular Crystallized Protein in Living Animal

by Masahiro Kuramochi, Ibuki Sugawara, Yoichi Shinkai, Kazuhiro Mio and Yuji C. Sasaki

Int. J. Mol. Sci. 2023, 24(23), 16914; https://doi.org/10.3390/ijms242316914 - 29 Nov 2023

Viewed by 648

Abstract

Understanding the cellular environment as molecular crowding that supports the structure-specific functional expression of biomolecules has recently attracted much attention. Time-resolved X-ray observations have the remarkable capability to capture the structural dynamics of biomolecules with subnanometre precision. Nevertheless, the measurement of the intracellular dynamics within live organisms remains a challenge. Here, we explore the potential of utilizing crystallized proteins that spontaneously form intracellular crystals to investigate their intracellular dynamics via time-resolved X-ray observations. We generated transgenic Caenorhabditis elegans specifically expressing the crystallized protein in cells and observed the formation of the protein aggregates within the animal cells. From the toxic-effect observations, the aggregates had minimal toxic effects on living animals. Fluorescence observations showed a significant suppression of the translational diffusion movements in molecules constituting the aggregates. Moreover, X-ray diffraction measurements provided diffraction signals originating from these molecules. We also observed the blinking behaviour of the diffraction spots, indicating the rotational motion of these crystals within the animal cells. A diffracted X-ray blinking (DXB) analysis estimated the rotational motion of the protein crystals on the subnanometre scale. Our results provide a time-resolved X-ray diffraction technique for the monitoring of intracellular dynamics. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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8 pages, 2763 KiB

Open AccessCommunication

The Blinking of Small-Angle X-ray Scattering Reveals the Degradation Process of Protein Crystals at Microsecond Timescale

by Tatsuya Arai, Kazuhiro Mio, Hiroki Onoda, Leonard M. G. Chavas, Yasufumi Umena and Yuji C. Sasaki

Int. J. Mol. Sci. 2023, 24(23), 16640; https://doi.org/10.3390/ijms242316640 - 23 Nov 2023

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Abstract

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal’s domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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15 pages, 5620 KiB

Open AccessArticle

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study

by Tsutomu Yamane, Takahiro Nakayama, Toru Ekimoto, Masao Inoue, Keigo Ikezaki, Hiroshi Sekiguchi, Masahiro Kuramochi, Yasuo Terao, Ken Judai, Minoru Saito, Mitsunori Ikeguchi and Yuji C. Sasaki

Int. J. Mol. Sci. 2023, 24(20), 15423; https://doi.org/10.3390/ijms242015423 - 21 Oct 2023

Viewed by 1085

Abstract

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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15 pages, 3545 KiB

Open AccessArticle

Molecular Dynamics Mappings of the CCT/TRiC Complex-Mediated Protein Folding Cycle Using Diffracted X-ray Tracking

by Kazutaka Araki, Takahiro Watanabe-Nakayama, Daisuke Sasaki, Yuji C. Sasaki and Kazuhiro Mio

Int. J. Mol. Sci. 2023, 24(19), 14850; https://doi.org/10.3390/ijms241914850 - 03 Oct 2023

Cited by 1 | Viewed by 1231

Abstract

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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12 pages, 2213 KiB

Open AccessCommunication

Ligand-Dependent Intramolecular Motion of Native Nicotinic Acetylcholine Receptors Determined in Living Myotube Cells via Diffracted X-ray Tracking

by Koichiro Oishi, Mayu Nagamori, Yasuhiro Kashino, Hiroshi Sekiguchi, Yuji C. Sasaki, Atsuo Miyazawa and Yuri Nishino

Int. J. Mol. Sci. 2023, 24(15), 12069; https://doi.org/10.3390/ijms241512069 - 28 Jul 2023

Cited by 2 | Viewed by 917

Abstract

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in signal transduction at the neuromuscular junction (NMJ). Movement of the nAChR extracellular domain following agonist binding induces conformational changes in the extracellular domain, which in turn affects the transmembrane domain and opens the ion channel. It is known that the surrounding environment, such as the presence of specific lipids and proteins, affects nAChR function. Diffracted X-ray tracking (DXT) facilitates measurement of the intermolecular motions of receptors on the cell membranes of living cells, including all the components involved in receptor function. In this study, the intramolecular motion of the extracellular domain of native nAChR proteins in living myotube cells was analyzed using DXT for the first time. We revealed that the motion of the extracellular domain in the presence of an agonist (e.g., carbamylcholine, CCh) was restricted by an antagonist (i.e., alpha-bungarotoxin, BGT). Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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Review

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18 pages, 14016 KiB

Open AccessReview

Diffracted X-ray Tracking for Observing the Internal Motions of Individual Protein Molecules and Its Extended Methodologies

by Yuji C. Sasaki

Int. J. Mol. Sci. 2023, 24(19), 14829; https://doi.org/10.3390/ijms241914829 - 02 Oct 2023

Viewed by 805

Abstract

In 1998, the diffracted X-ray tracking (DXT) method pioneered the attainment of molecular dynamics measurements within individual molecules. This breakthrough revolutionized the field by enabling unprecedented insights into the complex workings of molecular systems. Similar to the single-molecule fluorescence labeling technique used in the visible range, DXT uses a labeling method and a pink beam to closely track the diffraction pattern emitted from the labeled gold nanocrystals. Moreover, by utilizing X-rays with extremely short wavelengths, DXT has achieved unparalleled accuracy and sensitivity, exceeding initial expectations. As a result, this remarkable advance has facilitated the search for internal dynamics within many protein molecules. DXT has recently achieved remarkable success in elucidating the internal dynamics of membrane proteins in living cell membranes. This breakthrough has not only expanded our knowledge of these important biomolecules but also has immense potential to advance our understanding of cellular processes in their native environment. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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11 pages, 1614 KiB

Open AccessReview

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

by Masayuki Oda

Int. J. Mol. Sci. 2023, 24(18), 13717; https://doi.org/10.3390/ijms241813717 - 06 Sep 2023

Cited by 1 | Viewed by 669

Abstract

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. Full article

(This article belongs to the Special Issue Single Molecule Tracking and Dynamics with Quantum Probes: New Advances and Challenges)

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