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1.
Small ; 20(25): e2400653, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38385848

ABSTRACT

Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.


Subject(s)
Amino Acids , Microscopy, Atomic Force , Peptides , Point Mutation , Peptides/chemistry , Amino Acids/chemistry , Surface Properties , Graphite/chemistry
2.
Bioinform Adv ; 4(1): vbad189, 2024.
Article in English | MEDLINE | ID: mdl-38205268

ABSTRACT

Motivation: Liquid-liquid phase separation (LLPS) enables compartmentalization in cells without biological membranes. LLPS plays essential roles in membraneless organelles such as nucleoli and p-bodies, helps regulate cellular physiology, and is linked to amyloid formation. Two types of proteins, scaffolds and clients, are involved in LLPS. However, computational methods for predicting LLPS client proteins from amino-acid sequences remain underdeveloped. Results: Here, we present Seq2Phase, an accurate predictor of LLPS client proteins. Information-rich features are extracted from amino-acid sequences by a deep-learning technique, Transformer, and fed into supervised machine learning. Predicted client proteins contained known LLPS regulators and showed localization enrichment into membraneless organelles, confirming the validity of the prediction. Feature analysis revealed that scaffolds and clients have different sequence properties and that textbook knowledge of LLPS-related proteins is biased and incomplete. Seq2Phase achieved high accuracies across human, mouse, yeast, and plant, showing that the method is not overfitted to specific species and has broad applicability. We predict that more than hundreds or thousands of LLPS client proteins remain undiscovered in each species and that Seq2Phase will advance our understanding of still enigmatic molecular and physiological bases of LLPS as well as its roles in disease. Availability and implementation: The software codes in Python underlying this article are available at https://github.com/IwasakiLab/Seq2Phase.

3.
RSC Adv ; 13(44): 30978-30984, 2023 Oct 18.
Article in English | MEDLINE | ID: mdl-37876657

ABSTRACT

Star polymers consisting of three helical poly(phenylacetylene) chains with a precisely controlled molecular weight (molar mass dispersity < 1.03) were successfully synthesized by the living polymerization of phenylacetylene derivatives with a Rh-based multicomponent catalyst system comprising trifunctional initiators, which have three phenylboronates centered on a benzene ring, the Rh complex [Rh(nbd)Cl]2, diphenylacetylene, triphenylphosphine, and a base. The analysis of chiroptical properties of the optically active star polymers obtained by the living polymerization of optically active phenylacetylene derivatives revealed that the star polymers exhibited chiral amplification properties owing to their unique topology compared with the corresponding linear polymers.

4.
Proc Natl Acad Sci U S A ; 120(36): e2217708120, 2023 09 05.
Article in English | MEDLINE | ID: mdl-37639600

ABSTRACT

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.


Subject(s)
Oryza , Oryza/genetics , Cytokinins/genetics , Purine Nucleosides , N-Glycosyl Hydrolases/genetics , Nucleosides , Cell Wall/genetics
6.
STAR Protoc ; 4(3): 102468, 2023 Sep 15.
Article in English | MEDLINE | ID: mdl-37481726

ABSTRACT

Atomic force microscopy (AFM) is capable of nanoscale imaging but has so far only been used on cell surfaces when applied to a living cell. Here, we describe a step-by-step protocol for nanoendoscopy-AFM, which enables the imaging of nanoscale structures inside living cells. The protocol consists of cell staining, fabrication of the nanoneedle probes, observation inside living cells using 2D and 3D nanoendoscopy-AFM, and visualization of the 3D data. For complete details on the use and execution of this protocol, please refer to Penedo et al. (2021)1 and Penedo et al. (2021).2.


Subject(s)
Nanotechnology , Microscopy, Atomic Force/methods , Nanotechnology/methods , Cell Membrane/chemistry
7.
Nanoscale Adv ; 4(23): 5027-5036, 2022 Nov 22.
Article in English | MEDLINE | ID: mdl-36504747

ABSTRACT

Stimuli-responsive polyelectrolyte brushes adapt their physico-chemical properties according to pH and ion concentrations of the solution in contact. We synthesized a poly(acrylic acid) bearing cysteine residues at side chains and a lipid head group at the terminal, and incorporated them into a phospholipid monolayer deposited on a hydrophobic silane monolayer. The ion-specific, nanoscale response of polyelectrolyte brushes was detected by using three-dimensional scanning force microscopy (3D-SFM) combined with frequency modulation detection. The obtained topographic and mechanical landscapes indicated that the brushes were uniformly stretched, undergoing a gradual transition from the brush to the bulk electrolyte in the absence of divalent cations. When 1 mM calcium ions were added, the brushes were uniformly compacted, exhibiting a sharper brush-to-bulk transition. Remarkably, the addition of 1 mM cadmium ions made the brush surface significantly rough and the mechanical landscape highly heterogeneous. Currently, cadmium-specific nanoscale compaction of the brushes is attributed to the coordination of thiol and carboxyl side chains with cadmium ions, as suggested for naturally occurring, heavy metal binding proteins.

8.
ACS Appl Mater Interfaces ; 14(39): 44947-44957, 2022 Oct 05.
Article in English | MEDLINE | ID: mdl-36126145

ABSTRACT

Anti-freezing surfactants form an adsorption layer at the solid-water interface to inhibit the nucleation and growth of ice. However, this mechanism has not been elucidated at the molecular scale because of the difficulties in visualizing such adsorption structures. In this study, we overcome this limitation by directly visualizing the three-dimensional (3D) adsorption structures of anti-freezing surfactants, hexadecyltrimethylammonium bromide (C16TABs), on sapphire (0001) surfaces through 3D scanning force microscopy. We present molecularly resolved two-dimensional/3D images of the adsorption structures in solutions of 1, 10, and 100 ppm. At 1 ppm, the molecules form a monolayer with a flat-lying configuration. At 10 ppm, the molecular orientation is closer to the upright configuration, with a relatively large tilt angle. At 100 ppm, the molecules form a bilayer with almost upright configurations, providing excellent screening of the sapphire surface from water. Owing to the steric and electrostatic repulsion between adjacent molecular head groups, the surface of the bilayer exhibits relatively large fluctuations, inhibiting the formation of stable ice-like structures. The understanding of molecular-level mechanisms provides important guidelines for improving the design of anti-freezing surfactants for practical applications such as car coolants.

9.
Small Methods ; 6(9): e2200320, 2022 09.
Article in English | MEDLINE | ID: mdl-35686343

ABSTRACT

Chitin is one of the most abundant and renewable natural biopolymers. It exists in the form of crystalline microfibrils and is the basic structural building block of many biological materials. Its surface crystalline structure is yet to be reported at the molecular level. Herein, atomic force microscopy (AFM) in combination with molecular dynamics simulations reveals the molecular-scale structural details of the chitin nanocrystal (chitin NC)-water interface. High-resolution AFM images reveal the molecular details of chitin chain arrangements at the surfaces of individual chitin NCs, showing highly ordered, stable crystalline structures almost free of structural defects or disorder. 3D-AFM measurements with submolecular spatial resolution demonstrate that chitin NC surfaces interact strongly with interfacial water molecules creating stable, well-ordered hydration layers. Inhomogeneous encapsulation of the underlying chitin substrate by these hydration layers reflects the chitin NCs' multifaceted surface character with different chain arrangements and molecular packing. These findings provide important insights into chitin NC structures at the molecular level, which is critical for developing the properties of chitin-based nanomaterials. Furthermore, these results will contribute to a better understanding of the chemical and enzymatic hydrolysis of chitin and other native polysaccharides, which is also essential for the enzymatic conversion of biomass.


Subject(s)
Nanoparticles , Water , Chitin , Microscopy, Atomic Force/methods , Polysaccharides , Water/chemistry
10.
Clin Exp Dent Res ; 8(5): 1124-1129, 2022 10.
Article in English | MEDLINE | ID: mdl-35719036

ABSTRACT

OBJECTIVES: The amount of propofol required for intravenous sedation (IVS) in patients on long-term oral benzodiazepine (BZD) therapy may be affected by drug interactions and central changes in sensitivity. However, there is no research on the effect of long-term oral BZD use on the amount of propofol required for IVS. We aimed to clarify the difference between the total propofol dose required for IVS in patients with or without long-term oral BZD therapy. MATERIAL AND METHODS: Among patients treated for 4 years, the total administered dose required for IVS with propofol alone and local anesthesia for the extraction of bilateral impacted mandibular wisdom teeth, was retrospectively compared between patients with continuous oral BZD use for ≥6 months (BZD group; n = 24) and those without such use (control group; n = 307). The aimed sedation level was the Ramsay sedation scale 3-4. RESULTS: The amount of propofol required for IVS was significantly lower in the BZD group compared to the control group (4.83 ± 1.30 vs. 5.91 ± 1.25 mg/kg/h, p < .001; 95% confidence interval, -1.22 to -0.94 mg/kg/h; Cohen's d, 0.84). The required propofol dose was not influenced by preoperative oral BZD administration on the day of extraction (presence [n = 13] vs. absence [n = 11]: 4.9 ± 1.3 vs. 4.8 ± 1.7 mg/kg/h, p = .83). Long-term oral BZD therapy remained a significant factor for a lower required propofol dose after adjusting for age with multiple linear regression analysis. The underlying mechanism cannot be an additive action process but might pertain to competitive inhibition via an enzyme involved in glucuronate conjugation or competitive albumin binding. CONCLUSIONS: Clinicians should understand that patients on long-term oral BZDs therapy might require less propofol for IVS than those not on BZDs, irrespective of whether BZDs were taken preoperatively on the day of surgery.


Subject(s)
Propofol , Albumins , Benzodiazepines/adverse effects , Conscious Sedation , Cross-Sectional Studies , Glucuronates , Humans , Propofol/therapeutic use , Retrospective Studies
11.
Commun Biol ; 5(1): 487, 2022 05 20.
Article in English | MEDLINE | ID: mdl-35595960

ABSTRACT

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.


Subject(s)
Artifacts , Membrane Proteins , Cell Membrane , Fixatives , Microscopy, Atomic Force
12.
Faraday Discuss ; 235(0): 551-561, 2022 07 14.
Article in English | MEDLINE | ID: mdl-35383345

ABSTRACT

We have investigated the calcite growth mechanism by directly imaging atomic-scale structural changes at the growing step edges with high-speed frequency modulation atomic force microscopy (HS-FM-AFM). We compared the results with those previously obtained during dissolution, where a transition region (TR) consisting of a Ca(OH)2 monolayer was found to be formed along the step edges as an intermediate state. We found that the TR is created not only during dissolution but also during the growth process. Steps with and without a TR coexist with a ratio of 7 : 3 in both dissolution and growth, implying that their primary reaction pathways should involve TR formation. While all the dissolving steps show a linear shape, the growing steps additionally present a complex non-linear shape with many kinks. The TRs formed along the linear steps present a fixed and uniform width, while those along the complex steps present a non-uniform and dynamically varying width. The acute and obtuse steps show similar TR formation probability, TR width, and step velocity during growth, while a TR is preferentially formed along an acute step during dissolution. For both step types, TRs during growth are wider than those during dissolution. Based on these findings, we present possible reaction pathways triggered by the adsorption of either CO2 or HCO3- for the elementary steps in calcite growth.


Subject(s)
Calcium Carbonate , Adsorption , Calcium Carbonate/chemistry , Microscopy, Atomic Force/methods
13.
Sci Adv ; 7(52): eabj4990, 2021 Dec 24.
Article in English | MEDLINE | ID: mdl-34936434

ABSTRACT

Atomic force microscopy (AFM) is the only technique that allows label-free imaging of nanoscale biomolecular dynamics, playing a crucial role in solving biological questions that cannot be addressed by other major bioimaging tools (fluorescence or electron microscopy). However, such imaging is possible only for systems either extracted from cells or reconstructed on solid substrates. Thus, nanodynamics inside living cells largely remain inaccessible with the current nanoimaging techniques. Here, we overcome this limitation by nanoendoscopy-AFM, where a needle-like nanoprobe is inserted into a living cell, presenting actin fiber three-dimensional (3D) maps, and 2D nanodynamics of the membrane inner scaffold, resulting in undetectable changes in cell viability. Unlike previous AFM methods, the nanoprobe directly accesses the target intracellular components, exploiting all the AFM capabilities, such as high-resolution imaging, nanomechanical mapping, and molecular recognition. These features should greatly expand the range of intracellular structures observable in living cells.

14.
Chem Commun (Camb) ; 57(92): 12266-12269, 2021 Nov 19.
Article in English | MEDLINE | ID: mdl-34704570

ABSTRACT

The helical structures of poly(diphenylacetylene)s bearing optically active substituents linked through amide bonds and with a helicity memory have been visualised using atomic force microscopy. The polymers self-assembled into an ordered 2D monolayer on highly oriented pyrolytic graphite upon exposure to solvent vapour, whose helical pitch and handedness (right- and left-handed) were for the first time directly revealed at molecular resolution.


Subject(s)
Acetylene/analogs & derivatives , Amides , Microscopy, Atomic Force , Solvents/chemistry , Stereoisomerism
15.
J Phys Chem Lett ; 12(33): 8039-8045, 2021 Aug 26.
Article in English | MEDLINE | ID: mdl-34402624

ABSTRACT

Calcite dissolution is initiated by the formation of a nanoscale etch pit followed by step edge propagation and hence strongly influenced by the interactions between surface diffusing ions and step edges. However, such atomic-scale dynamics are mostly inaccessible with current imaging tools. Here, we overcome this limitation by using our recent development of high-speed frequency modulation atomic force microscopy. By visualizing atomic-scale structural changes of the etch pits at the calcite surface in water, we found the existence of mobile and less-mobile surface adsorption layers (SALs) in the etch pits. We also found that some etch pits maintain their size for a long time without expansion, and their step edges are often associated with less-mobile SALs, suggesting their step stabilization effect.


Subject(s)
Calcium Carbonate/chemistry , Microscopy, Atomic Force/methods , Nanostructures/chemistry , Adsorption , Crystallography , Molecular Structure , Solubility , Surface Properties , Water/chemistry
16.
Phys Chem Chem Phys ; 20(36): 23522-23527, 2018 Sep 19.
Article in English | MEDLINE | ID: mdl-30183018

ABSTRACT

Investigating interfacial water ordering on solid surfaces with different hydrophobicities is fundamentally important. Here, we prepared hydrophilic mica substrates with some areas covered by mildly hydrophobic graphene layers and studied the resulting hydration layers using three-dimensional (3D) force measurements based on frequency-modulation atomic force microscopy. Hydration layers of 0.3-0.6 nm were detected on bare graphene regions; these layers were considerably larger than the spacing measured on mica (0.2-0.3 nm). On the graphene-covered regions, we also observed the formation of special ordered structures of adsorbates over time, on which, surprisingly, no prominent hydration layers were detected. Based on these findings, we present one possible scenario to describe the formation process of the ordered interfacial structures and the enhanced oscillation period in the force profiles. This work also demonstrates the capability and significance of 3D force measurements in probing hydration behaviors on a heterogeneous substrate with a lateral resolution smaller than several nanometers.

17.
Beilstein J Nanotechnol ; 9: 1844-1855, 2018.
Article in English | MEDLINE | ID: mdl-30013878

ABSTRACT

A phase-locked loop (PLL) circuit is the central component of frequency modulation atomic force microscopy (FM-AFM). However, its response speed is often insufficient, and limits the FM-AFM imaging speed. To overcome this issue, we propose a PLL design that enables high-speed FM-AFM. We discuss the main problems with the conventional PLL design and their possible solutions. In the conventional design, a low-pass filter with relatively high latency is used in the phase feedback loop, leading to a slow response of the PLL. In the proposed design, a phase detector with a low-latency high-pass filter is located outside the phase feedback loop, while a subtraction-based phase comparator with negligible latency is located inside the loop. This design minimizes the latency within the phase feedback loop and significantly improves the PLL response speed. In addition, we implemented PLLs with the conventional and proposed designs in the same field programmable gate array chip and quantitatively compared their performances. The results demonstrate that the performance of the proposed PLL is superior to that of the conventional PLL: 165 kHz bandwidth and 3.2 µs latency in water. Using this setup, we performed FM-AFM imaging of calcite dissolution in water at 0.5 s/frame with true atomic resolution. The high-speed and high-resolution imaging capabilities of the proposed design will enable a wide range of studies to be conducted on various atomic-scale dynamic phenomena at solid-liquid interfaces.

18.
Phys Rev Lett ; 120(11): 116101, 2018 Mar 16.
Article in English | MEDLINE | ID: mdl-29601750

ABSTRACT

It seems natural to assume that defects at mineral surfaces critically influence interfacial processes such as the dissolution and growth of minerals in water. The experimental verification of this claim, however, is challenging and requires real-space methods with utmost spatial resolution, such as atomic force microscopy (AFM). While defects at mineral-water interfaces have been resolved in 2D AFM images before, the perturbation of the surrounding hydration structure has not yet been analyzed experimentally. In this Letter, we demonstrate that point defects on the most stable and naturally abundant calcite (10.4) surface can be resolved using high-resolution 3D AFM-even within the fifth hydration layer. Our analysis of the hydration structure surrounding the point defect shows a perturbation of the hydration with a lateral extent of approximately one unit cell. These experimental results are corroborated by molecular dynamics simulations.

19.
Nano Lett ; 17(7): 4083-4089, 2017 07 12.
Article in English | MEDLINE | ID: mdl-28650174

ABSTRACT

The microscopic understanding of the crystal growth and dissolution processes have been greatly advanced by the direct imaging of nanoscale step flows by atomic force microscopy (AFM), optical interferometry, and X-ray microscopy. However, one of the most fundamental events that govern their kinetics, namely, atomistic events at the step edges, have not been well understood. In this study, we have developed high-speed frequency modulation AFM (FM-AFM) and enabled true atomic-resolution imaging in liquid at ∼1 s/frame, which is ∼50 times faster than the conventional FM-AFM. With the developed AFM, we have directly imaged subnanometer-scale surface structures around the moving step edges of calcite during its dissolution in water. The obtained images reveal that the transition region with typical width of a few nanometers is formed along the step edges. Building upon insight in previous studies, our simulations suggest that the transition region is most likely to be a Ca(OH)2 monolayer formed as an intermediate state in the dissolution process. On the basis of this finding, we improve our understanding of the atomistic dissolution model of calcite in water. These results open up a wide range of future applications of the high-speed FM-AFM to the studies on various dynamic processes at solid-liquid interfaces with true atomic resolution.

20.
Nanotechnology ; 27(41): 415709, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27609045

ABSTRACT

Frequency modulation atomic force microscopy (FM-AFM) experiments were performed on the calcite (10[Formula: see text]4) surface in pure water, and a detailed analysis was made of the 2D images at a variety of frequency setpoints. We observed eight different contrast patterns that reproducibly appeared in different experiments and with different measurement parameters. We then performed systematic free energy calculations of the same system using atomistic molecular dynamics to obtain an effective force field for the tip-surface interaction. By using this force field in a virtual AFM simulation we found that each experimental contrast could be reproduced in our simulations by changing the setpoint, regardless of the experimental parameters. This approach offers a generic method for understanding the wide variety of contrast patterns seen on the calcite surface in water, and is generally applicable to AFM imaging in liquids.

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