In-Cell NMR in Xenopus laevis Oocytes

Thongwichian, Rossukon; Selenko, Philipp

doi:10.1007/978-1-61779-927-3_3

Cited by 12 publications

(24 citation statements)

References 18 publications

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“…These results show that the phosphorylation state of the UD of Src represents a sensor of the kinase-phosphatase network active at any given moment in the cell. Experimental access to this information is possible by using real-time NMR techniques (Selenko et al, 2008; Theillet et al, 2012; Thongwichian and Selenko, 2012; Amata et al, 2013). In contrast to folded domains, intrinsically disordered domains, like the UD of Src, provide in vivo NMR resolution comparable to that obtainable in vitro (Kosol et al, 2013).…”

Section: Phosphorylation Of the Unique Domain Of Srcmentioning

confidence: 99%

Phosphorylation of unique domains of Src family kinases

2014

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Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered “Unique” domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes.

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Section: Phosphorylation Of the Unique Domain Of Srcmentioning

confidence: 99%

Phosphorylation of unique domains of Src family kinases

2014

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“…Protein-protein interactions [ 173 ] and the behavior of intrinsically disordered proteins [ 174 , 175 ] can be evaluated in cells using this approach. In eukaryotic cells, the first in-cell NMR experiments were carried out on labeled proteins that were injected into the oocytes of Xenopus laevis [ 7 , 176 , 177 ]. Using cell-penetrating peptides, an isotopically labeled protein can be delivered into living human cells.…”

Section: Solution Nmr In Target Engagementmentioning

confidence: 99%

Solution NMR Spectroscopy in Target-Based Drug Discovery

Kang

2017

Molecules

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Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more and more important roles in multiple steps of the drug discovery process. In a fragment-based drug discovery process, ligand-observed and protein-observed NMR spectroscopy can be applied to screen fragments with low binding affinities. The screened fragments can be further optimized into drug-like molecules. In combination with other biophysical techniques, NMR will guide structure-based drug discovery. In this review, we describe the possible roles of NMR spectroscopy in drug discovery. We also illustrate the challenges encountered in the drug discovery process. We include several examples demonstrating the roles of NMR in target-based drug discoveries such as hit identification, ranking ligand binding affinities, and mapping the ligand binding site. We also speculate the possible roles of NMR in target engagement based on recent processes in in-cell NMR spectroscopy.

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“…Detailed protocols for the use of many different cell types, including bacteria, mammalian cells, and other eukaryotic cells, have been developed and established. [12,13,[102][103][104][105] The advantage of the low energy needed for excitation is at the same time, however, also the biggest disadvantage of NMR spectroscopy. The low excitation energy correlates with a low energy gap between the ground and the excited state and therefore only a small population excess in the ground state, thus leading to the inherently low sensitivity of the method.…”

Section: Future Perspectives Of In-cell Nmr Spectroscopymentioning

confidence: 99%

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

et al. 2014

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The dream of cell biologists is to be able to watch biological macromolecules perform their duties in the intracellular environment of live cells. Ideally, the observation of both the location and the conformation of these macromolecules with biophysical techniques is desired. The development of many fluorescence techniques, including superresolution fluorescence microscopy, has significantly enhanced our ability to spot proteins and other molecules in the crowded cellular environment. However, the observation of their structure and conformational changes while they attend their business is still very challenging. In principle, NMR and EPR spectroscopy can be used to investigate the conformation and dynamics of biological macromolecules in living cells. The development of in‐cell magnetic resonance techniques has demonstrated the feasibility of this approach. Herein we review the different techniques with a focus on liquid‐state in‐cell NMR spectroscopy, provide an overview of applications, and discuss the challenges that lie ahead.

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In-Cell NMR in Xenopus laevis Oocytes

Cited by 12 publications

References 18 publications

Phosphorylation of unique domains of Src family kinases

Phosphorylation of unique domains of Src family kinases

Solution NMR Spectroscopy in Target-Based Drug Discovery

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

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