Enzymatic Labeling of Arbitrary Proteins

Shimba, Nobuhisa; Yamada, Naoyuki; Yokoyama, Keiichi; Suzuki, Eiichiro

doi:10.1006/abio.2001.5485

Cited by 19 publications

(10 citation statements)

References 27 publications

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“…The 1 H- 15 N correlation signal of the side chain carboxyamide group of glutamine and asparagine residues is useful as an NMR probe, since it is sharper than that of the backbone amide signals and is applicable to large molecular weight proteins. For the γ-carboxyamide group of the glutamine residue, enzymatic 15 N-incorporation is achieved using recombinant protein transglutaminase (TGase) [ 142 , 143 ]. The TGase catalyzes the chemical replacement of the γ-carboxyamide group with free ammonium ions, under mild reaction conditions without structural changes, undesired degradation, or precipitation.…”

Section: Isotope Labeling Of Target Proteins For Drug Discovery Bymentioning

confidence: 99%

Current NMR Techniques for Structure-Based Drug Discovery

et al. 2018

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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.

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Section: Isotope Labeling Of Target Proteins For Drug Discovery Bymentioning

confidence: 99%

Current NMR Techniques for Structure-Based Drug Discovery

et al. 2018

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“…In order to optimize this process, it would be desirable to have a rapid and efficient method for screening target ORFs for their suitability for structural studies. Several methods have been developed to screen candidates for structural studies, such as cell free expression systems [20,21] and enzymatic labeling of glutamine in vitro [22] and selective isotopic labeling of the target proteins in vivo [23].…”

Section: Introductionmentioning

confidence: 99%

High‐throughput screening of structural proteomics targets using NMR

et al. 2003

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We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid 1 H^1 5 N HSQC NMR analysis of bacterial lysates containing selectively 15 N-labelled heterologous proteins. Our analysis permitted us to classify the 19 soluble candidates in terms of 'foldedness', that is, the extent to which they present a well-folded solution structure, as re£ected by the quality of their NMR spectra. This classi¢cation allowed us to de¢ne a priority list to be used as a guide to select protein candidates for further structural studies. ß

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“…Despite many effective methods being available for the incorporation of radioisotopes into proteins [23][24][25][26][27][28][29], radioisotope-labeled proteins are rarely used in EMSA. Nucleic acid labeling procedures are more convenient and provide higher specific activities, which results in a more sensitive detection than with comparable proteinlabeling reactions.…”

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confidence: 99%

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Fried

2012

Alternative Pre‐mRNA Splicing

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The electrophoretic mobility shift assay (EMSA) is commonly used to study proteinnucleic acid interactions. The technique is based on the observation that proteinbound nucleic acid molecules migrate more slowly than free nucleic acid molecules when subjected to native polyacrylamide or agarose gel electrophoresis. After electrophoresis, the distribution of species containing nucleic acid is determined by autoradiography or other sensitive imaging technique. Under appropriate conditions, the method can be used for quantitative binding analysis, but it is most widely used as a qualitative means of detecting protein-RNA complexes. In this chapter, features relevant to the design of EMSA experiments are identified, and the technical factors found to be important for the success of the assay are outlined. Theoretical BackgroundThe electrophoretic mobility shift assay (EMSA) is a simple and powerful method for detecting interactions between nucleic acids and proteins [1][2][3][4][5][6]. Although the simplicity of the method accounts for its popularity, it also conceals technical subtleties. In this chapter, the aim is to help select the most convenient EMSA variants for experimental purposes, and to avoid the most important pitfalls associated with the technique.The EMSA is based on the observation that the gel-electrophoretic mobility of a protein-nucleic acid complex is often less than that of the corresponding free nucleic acid. Current versions of the assay differ little from those originally described by Garner and Revzin [7] and Fried and Crothers [8], although precursors can be found in earlier reports [9][10][11]. Although originally developed to analyze DNA-protein complexes, the method is also useful for the characterization of RNA-protein interactions [12][13][14].The electrophoretic mobilities of RNA molecules in polyacrylamide or agarose gels depend on: (i) the sizes, shapes, and charges of the molecules; (ii) the conductivities of the gel and sample buffers; and (iii) the concentrations of the gel polymer and its degree of crosslinking [15]. Protein binding can result in complexes that differ from the parent RNA in terms of charge, size or shape; changes in any or all of these factors may produce a difference in gel-mobility that allows the detection of binding. Changes in the type of gel matrix (e.g., agarose versus polyacrylamide), its concentration, and changes in the composition of the gel buffer, can each affect electrophoretic resolution and, just as importantly, the stabilities of the protein-nucleic acid complexes within the gel [16][17][18][19]. Optimization of these factors is straightforward and can significantly improve the results of an EMSA.Alternative pre-mRNA Splicing: Theory and Protocols, First Edition. EditedThe selection of a binding substrate requires assumptions to be made about the RNA structure(s) needed for appropriate protein binding. If the structure of the RNA probe differs significantly from the native cellular binding target, the behavior of the assay system may not be repre...

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Enzymatic Labeling of Arbitrary Proteins

Cited by 19 publications

References 27 publications

Current NMR Techniques for Structure-Based Drug Discovery

Current NMR Techniques for Structure-Based Drug Discovery

High‐throughput screening of structural proteomics targets using NMR

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

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