The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy.

Cadherin EGF LAG seven-pass G-type receptors (CELSR) cadherins, members of the cadherin superfamily, and adhesion G-protein-coupled receptors, play a vital role in cell-cell adhesion. The mutual binding of the extracellular domains (ectodomains) of CELSR cadherins between cells is crucial for tissue formation, including the establishment of planar cell polarity, which directs the proper patterning of cells. CELSR cadherins possess nine cadherin ectodomains (EC1-EC9) and noncadherin ectodomains. However, the structural and functional mechanisms of the binding mode of CELSR cadherins have not been determined. In this study, we investigated the binding mode of CELSR cadherins using single-molecule fluorescence microscopy, high-speed atomic force microscopy (HS-AFM), and bead aggregation assay. The fluorescence microscopy analysis results indicated that the trans-dimer of the CELSR cadherin constitutes the essential adhesive unit between cells. HS-AFM analysis and bead aggregation assay results demonstrated that EC1-EC8 entirely overlap and twist to form antiparallel dimer conformations and that the binding of EC1-EC4 is sufficient to sustain bead aggregation. The interaction mechanism of CELSR cadherin may elucidate the variation of the binding mechanism within the cadherin superfamily and physiological role of CELSR cadherins in relation to planar cell polarity.

Protein degradation by a component of the chaperonin-linked protease ClpP.

In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.

Identification of potential C1-binding sites in the immunoglobulin CL domains.

Immunoglobulin G (IgG) molecules that bind antigens on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single-molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centred on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

The two-step cargo recognition mechanism of heterotrimeric kinesin.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy.

Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

In Situ Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope.

High-speed atomic force microscopy (HS-AFM) is an indispensable technique in the field of biology owing to its imaging capability with high spatiotemporal resolution. Furthermore, recent developments established tip-scan stand-alone HS-AFM combined with an optical microscope, drastically improving its versatility. It has considerable potential to contribute to not only biology but also various research fields. A great candidate is a photoactive material, such as an azo-polymer, which is important for optical applications because of its unique nanoscale motion under light irradiation. Here, we demonstrate the in situ observation of nanoscale azo-polymer motion by combining tip-scan HS-AFM with an optical system, allowing HS-AFM observations precisely aligned with a focused laser position. We observed the dynamic evolution of unique morphologies in azo-polymer films. Moreover, real-time topographic line profile analyses facilitated precise investigations of the morphological changes. This important demonstration would pave the way for the application of HS-AFM in a wide range of research fields.

Photoresponsive Supramolecular Polymers Capable of Intrachain Folding and Interchain Aggregation.

The competition between polymer chain folding and aggregation is a critical structuring process that determines the physical properties of synthetic and biopolymers. However, supramolecular polymer systems that exhibit both processes have not yet been reported. We herein introduce a system in which folded supramolecular polymers spontaneously undergo interchain aggregation due to a rearrangement in internal molecular order, converting them into crystalline aggregates. These folded supramolecular polymers slowly crystallize over the course of half a day, due to their characteristic higher-order structures. However, the photoisomerization of the trans-azobenzene incorporated into the monomer to the cis isomer leads to unfolding of the polymer, accelerating the intrachain and interchain molecular ordering to a few hours. The intermediate structures visualized by AFM demonstrate that the unfolding is coupled with interchain aggregation.

Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid ß Fibrils.

Understanding the dynamic assembly process of amyloid ß (Aß) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aß fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aß antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aß monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aß assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product.

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Nanoscale Structures of Tough Microparticle-Based Films Investigated by Synchrotron X-Ray Scattering and All-Atom Molecular-Dynamics Simulation.

In this study, the nanoscale structures of microparticle-based films are revealed by synchrotron small-angle X-ray scattering (SAXS) and all-atom molecular-dynamics (AA-MD) simulations. The microparticle-based films consisting of the simplest acrylate polymer microparticles are applied as a model because the films are formed without additives and organic solvents and exhibit high toughness properties. The characteristic interfacial thickness (tinter) obtained from the SAXS analysis reflects the mixing degree of polymer chains on the microparticle surface in the film. The cross-linking density of inner microparticles is found to be strongly correlated to not only several properties of individual microparticles, such as swelling ratio and radius of gyration, but also the tinter and toughness of the corresponding films. Therefore, the tinter and toughness values follow a linear relationship because the cross-linking restricts the mixing of polymer chains between their surfaces in the film, which is a unique feature of microparticle-based films. This characteristic also affects their deformation behavior observed by in situ SAXS during tensile testing and their density profiles calculated by AA-MD simulations. This work provides a general strategy for material design to control the physical properties and structures of their films for advanced applications, including volatile organic compound-free sustainable coatings and adhesives.

How Resilient is Wood Xylan to Enzymatic Degradation in a Matrix with Kraft Lignin?

Despite the potential of lignocellulose in manufacturing value-added chemicals and biofuels, its efficient biotechnological conversion by enzymatic hydrolysis still poses major challenges. The complex interplay between xylan, cellulose, and lignin in fibrous materials makes it difficult to assess underlying physico- and biochemical mechanisms. Here, we reduce the complexity of the system by creating matrices of cellulose, xylan, and lignin, which consists of a cellulose base layer and xylan/lignin domains. We follow enzymatic degradation using an endoxylanase by high-speed atomic force microscopy and surface plasmon resonance spectroscopy to obtain morphological and kinetic data. Fastest reaction kinetics were observed at low lignin contents, which were related to the different swelling capacities of xylan. We demonstrate that the complex processes taking place at the interfaces of lignin and xylan in the presence of enzymes can be monitored in real time, providing a future platform for observing phenomena relevant to fiber-based systems.

Elastomer Particle Monolayers Formed by the Compression of Poly(methyl acrylate) Microparticles at an Air/Water Interface.

In the previous study (Green Chem., 2023, 25, 3418), highly stretchable and mechanically tough poly(methyl acrylate) (pMA) microparticle-based elastomers can be formed by drying a microparticle-containing aqueous dispersion. This discovery has the potential to overcome the mechanical weakness of industrially produced aqueous latex films. However, in 3D-arranged particle films, structural complexity, such as the existence of defects, makes it difficult to clearly understand the relationship between the particle film structure and its mechanical properties. In this study, 2D-ordered pMA particle monolayers at the air/water interface of a Langmuir trough are prepared. Under high compression at the air/water interface, the microparticles contact their neighboring particles, and the resulting monolayers can be successfully transferred onto a solid substrate. The compression of the monolayer films is linked to an increase in the elastic modulus of the monolayer film on the solid substrate as evident from the local Young's modulus mapping using atomic force microscopy. Thus, pMA particle films with different mechanical properties can be created using a Langmuir trough.

Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy.

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

Single microgel degradation governed by heterogeneous nanostructures.

Although the degradation of colloidal particles is one of the most attractive phenomena in the field of biological and environmental science, the degradation mechanism of single particles remains to be elucidated. In this study, in order to clarify the impact of the structure of a single particle on the oxidative degradation processes, thermoresponsive colloidal particles with chemical cleavage points were synthesized as a model, and their degradation behavior was evaluated using high-speed atomic force microscopy (HS-AFM) as well as conventional scattering techniques. The real-time observation of single-particle degradation revealed that the degradation behavior of microgels is governed by their inhomogeneous nanostructure, which originates from the polymerization method and their hydrophilicity. Our findings can be expected to advance the design of carriers for drug-delivery and the understanding of the formation processes of micro (nano)plastics.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19 F†MR Imaging.

19 F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19 F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19 F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19 F MRI imaging. Importantly, our 19 F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.