39 research outputs found
NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase
Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases
In-cell NMR as a sensitive tool to monitor physiological condition of Escherichia coli
Sugiki, T., Yamaguchi, Y., Fujiwara, T. et al. In-cell NMR as a sensitive tool to monitor physiological condition of Escherichia coli. Sci Rep 10, 2466 (2020). https://doi.org/10.1038/s41598-020-59076-2
The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase
This research was originally published in Journal of Biological Chemistry. Ken-ichi Kosami, Izuru Ohki, Minoru Nagano, Kyoko Furuita, Toshihiko Sugiki, Yoji Kawano, Tsutomu Kawasaki, Toshimichi Fujiwara, Atsushi Nakagawa, Ko Shimamoto and Chojiro Kojima. The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase. Journal of Biological Chemistry. 2014; 289, 28569-28578. Ā© the American Society for Biochemistry and Molecular Biology
An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function
NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication
Site-specific isotope labeling of long RNA for structural and mechanistic studies
A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, āisotopically labeled guanosine 5ā²-monophosphateā (5ā²-GMP), steadily gave a 5ā²-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5ā²-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry
Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.
Although electrostatic interactions between negatively-charged ferredoxin (Fd) and positively-charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We herein investigated interprotein forces for formation of an electron-transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum around 40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay . Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on addition of SiR to 15N-labeled Fd at the various NaCl concentration also demonstrated that a combination of electro- and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions
Current NMR Techniques for Structure-Based Drug Discovery
A variety of nuclear magnetic resonance (NMR) applications have been developed for structure-based drug discovery (SBDD). NMR provides many advantages over other methods, such as the ability to directly observe chemical compounds and target biomolecules, and to be used for ligand-based and protein-based approaches. NMR can also provide important information about the interactions in a protein-ligand complex, such as structure, dynamics, and affinity, even when the interaction is too weak to be detected by ELISA or fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) or to be crystalized. In this study, we reviewed current NMR techniques. We focused on recent progress in NMR measurement and sample preparation techniques that have expanded the potential of NMR-based SBDD, such as fluorine NMR (19F-NMR) screening, structure modeling of weak complexes, and site-specific isotope labeling of challenging targets
Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks
Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks.
Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90ā95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis.
Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html.ISSN:1367-4803ISSN:1460-205