Chemical labelling for visualizing native AMPA receptors in live neurons

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders

Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins

Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research

Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni²⁺-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni²⁺–NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability

Electron microscopic detection of single membrane proteins by a specific chemical labeling

Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

ホルマリン漬けから着想した小分子可視化法 --医薬品開発効率化につながる新たな戦略--. 京都大学プレスリリース. 2022-12-05.Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed “fixation-driven chemical cross-linking of exogenous ligands (FixEL), ” which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules

人工電子チャネルの構築と機能

京都大学0048新制・課程博士工学博士甲第4081号工博第1041号新制||工||737(附属図書館)UT51-63-P21京都大学大学院工学研究科合成化学専攻(主査)教授 松浦 輝男, 教授 庄野 達哉, 教授 吉田 善一学位規則第5条第1項該当Kyoto UniversityDFA

Microscopic Imaging Techniques for Molecular Assemblies: Electron, Atomic Force, and Confocal Microscopies

Self-assembly is promising for construction of a wide variety of supramolecular assemblies, whose 1D/2D/3D structures are typically relevant to their functions. In-depth understanding of their structure–function relationships is essential for rational design and development of functional molecular assemblies. Microscopic imaging has been used as a powerful tool to elucidate structures of individual molecular assemblies with subnanometer to millimeter resolution, which is complementary to conventional spectroscopic techniques that provide the ensemble structural information. In this review, we highlight the representative examples of visualization of molecular assemblies by use of electron microscopy, atomic force microscopy, confocal microscopy, and super-resolution microscopy. This review comprehensively describes imaging of supramolecular nanofibers/gels, micelles/vesicles, coacervate droplets, polymer assemblies, and protein/DNA assemblies. Advanced imaging techniques that can address key challenges, like evaluation of dynamics of molecular assemblies, multicomponent self-assembly, and self-assembly/disassembly in complex cellular milieu, are also discussed. We believe this review would provide guidelines for deeper structural analyses of molecular assemblies to develop the next-generation materials

Recent applications of N-acyl imidazole chemistry in chemical biology

N-Acyl imidazoles are unique electrophiles that exhibit moderate reactivity, relatively long-half life, and high solubility in water. Thanks to their tunable reactivity and chemical selectivity, the application of N-acyl imidazole derivatives has launched to a number of chemical biology researches, which include chemical synthesis of peptide/protein, chemical labeling of native proteins of interest (POIs), and structural analysis and functional manipulation of RNAs. Since proteins and RNAs play pivotal roles in numerous biological events in all living organisms, the methods that enable the chemical modification of endogenously existing POIs and RNAs in live cells may offer a variety of opportunities not only for fundamental scientific study but also for biotechnology and drug development. In this review, we discuss the recent progress of N-acyl imidazole chemistry that contributes to the chemical labeling and functional control of endogenous proteins and RNAs under multimolecularly crowded biological conditions of live cells

Cell‐Like Synthetic Supramolecular Soft Materials Realized in Multicomponent, Non‐/Out‐of‐Equilibrium Dynamic Systems

Abstract Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio‐supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. These biological design strategies offer valuable insights for the development of synthetic supramolecular systems with spatially controlled hierarchical structures, which, importantly, exhibit cell‐like responses and functions. The next grand challenge in supramolecular chemistry is to control the organization of multiple types of supramolecules in a single system, thus integrating the functions of these supramolecules in an orthogonal and/or cooperative manner. In this perspective, the recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self‐assembled nanofibers/gels and coacervates, with other functional molecules, including polymer gels and enzymes is highlighted. Moreover, results show that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double‐network hydrogels, temporal stimulus pattern‐dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction–diffusion processes are presented. Autonomous active soft materials with cell‐like responses and spatially controlled structures hold promise for diverse applications, including soft robotics with directional motion, point‐of‐care disease diagnosis, and tissue regeneration