Rossukon Thongwichian

Institute of Molecular Biology, Mayence, Rheinland-Pfalz, Germany

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Publications (4)17.16 Total impact

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    ABSTRACT: We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the (15)N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D (1)H-(15)N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.
    Nature Protocol 06/2013; 8(7):1416-1432. · 8.36 Impact Factor
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    ABSTRACT: We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments.
    Biochimica et Biophysica Acta 01/2013; · 4.66 Impact Factor
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    ABSTRACT: Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.
    Journal of Biomolecular NMR 09/2012; · 2.85 Impact Factor
  • Rossukon Thongwichian, Philipp Selenko
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    ABSTRACT: For the purpose of studying IDPs inside cells of higher organisms, several eukaryotic in-cell NMR systems have been developed over the past years. In this chapter we will focus on high-resolution in-cell NMR applications in Xenopus laevis oocytes, the first eukaryotic cellular model system to be established. In contrast to prokaryotic in-cell NMR samples, eukaryotic in-cell NMR specimens are prepared by cytoplasmic delivery of an exogenously produced, isotope-labeled protein into the non-isotope-labeled environment of the respective "host" cell. In-cell NMR applications in Xenopus oocytes rely on intracellular sample deposition by direct microinjection into the oocyte cytoplasm. Here, we describe the preparation of oocyte in-cell NMR samples for IDP studies in this cellular model environment.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 895:33-41. · 1.29 Impact Factor