An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells

Hembram, Dambarudhar Shiba Sankar; Haremaki, Takahiro; Hamatsu, Jumpei; Inoue, Jin; Kamoshida, Hajime; Ikeya, Teppei; Mishima, Masaki; Mikawa, Tsutomu; Hayashi, Nobuhiro; Shirakawa, Masahiro; Ito, Yutaka

doi:10.1016/j.bbrc.2013.07.127

Cited by 27 publications

(32 citation statements)

References 41 publications

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“…In both cases, well-resolved 3D 13 C-separated NOESY spectra were acquired, indicating that our approach is effective for NOEbased structural analysis of proteins with molecular weight over 15 kDa in eukaryotic cells.F urthermore,c omparison of in-cell NMR spectra of CaM with corresponding spectra in diluted solution ( Figure 5A;S upporting Information, Figure S10) suggest that CaM in sf9 cells exists in astate similar to Mg 2+ -bound CaM in diluted solution. [15] In-cell NMR spectra suggest that HRas in sf9 cells is in the "inactive" guanosine diphosphate (GDP)-bound state ( Figure 5B;S upporting Information, Figure S11), which is reasonable considering that the C-terminal truncation prohibits the GDP to guanosine triphosphate (GTP) exchange at the cell membrane,w hile HRas-bound GTP will be hydrolyzed by its intrinsic GTPase activity. [15] In-cell NMR spectra suggest that HRas in sf9 cells is in the "inactive" guanosine diphosphate (GDP)-bound state ( Figure 5B;S upporting Information, Figure S11), which is reasonable considering that the C-terminal truncation prohibits the GDP to guanosine triphosphate (GTP) exchange at the cell membrane,w hile HRas-bound GTP will be hydrolyzed by its intrinsic GTPase activity.…”

Section: Angewandte Chemiementioning

confidence: 77%

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High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

et al. 2019

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Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions,itisindispensable to observe their threedimensional (3D) structures at the atomic level in aphysiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/ baculovirus system using NMR data from living cells exclusively.The method was applied to five proteins,rat calmodulin, human HRas,h uman ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein GB 1d omain. In all cases,w ec ould obtain structural information from wellresolved in-cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be astandard tool for protein structure determinations in living eukaryotic cells.F or three proteins,w ea chieved well-converged 3D structures.A mong these,t he in-cell structure of protein GB 1 domain was most accurately determined, demonstrating that ah elix-loop region is tilted away from a b-sheet compared to the conformation in diluted solution.Biomacromolecules occupy as ignificant fraction of the intracellular volume (resulting in molecular crowding) [1] in which proteins are exposed to the excluded-volume effect, specific and non-specific interactions,a nd various dynamic intracellular processes. [2] Their biophysical properties under these effects,p articularly their molecular structures at the atomic level, are not fully understood. Therefore,i ti s indispensable to elucidate their native structures and dynamics in the physiologically natural environment inside cells,and to determine whether there are differences in the threedimensional (3D) structures of the biomacromolecules in cells compared to their diluted solution state.I n-cell NMR [3] is currently the only tool with which to observe proteins and deoxyribonucleic acid (DNA) at atomic resolution in living biological systems.I ta lso provides direct observations of protein behaviors in conjunction with chemical compounds that are potential targets for drug screening inside cells. [2a,4] Although in-cell NMR studies in various eukaryotic cells have become possible by either expressing target proteins inside cells [4] or by introducing stable isotope-enriched proteins from outside, [2a, 5] high-resolution protein 3D structures have been determined only in Escherichia coli cells. [6] To date,t he achievable target-protein concentration in eukaryotic cells was too low to obtain as ufficient number of nuclear Overhauser effect (NOE)-derived distance restraints.I nt he meantime,in-cell NMR studies of human-cultured cells have revealed that the intracellular environment does indeed influence the protein folding stability [5f] and reduces the volume occupied by intrinsically disordered proteins. [7] Recently,p rotein global folds in cells were obtained by exploiting NMR chemical shifts and paramagnetic NMR effects induced by intracellularly stable lanthanoid-binding t...

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Section: Angewandte Chemiementioning

confidence: 77%

“…1 H-1 Hcross sections extracted from the 3D 13 C-(left) and15 N-separated (right) NOESY spectra of GB1 in living sf9 cells. Manually assigned peaks are labeled for intra-(blue) and inter-residue (red) NOEs.…”

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confidence: 99%

High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

et al. 2019

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“…As such, the living organism becomes the “ultimate biosensor” responding in real‐time to its environment, while the NMR spectrometer interprets the biochemical changes, providing information explaining sublethal toxicity at the molecular level. The reproducibility of NMR is second to none, making it a versatile profiling tool for the identification of stressed or diseased individuals . The manifestation of physiological responses to the exposure of toxins in the metabolic profile is rapid, compared with the genome or proteome, making the metabolome a key indicator of stress.…”

Section: Introductionmentioning

confidence: 99%

Flow‐based in vivo NMR spectroscopy of small aquatic organisms

Soong

Mobarhan

Tabatabaei

et al. 2019

Magnetic Reson in Chemistry

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NMR applied to living organisms is arguably the ultimate tool for understanding environmental stress responses and can provide desperately needed information on toxic mechanisms, synergistic effects, sublethal impacts, recovery, and biotransformation of xenobiotics. To perform in vivo NMR spectroscopy, a flow cell system is required to deliver oxygen and food to the organisms while maintaining optimal line shape for NMR spectroscopy. In this tutorial, two such flow cell systems and their constructions are discussed: (a) a single pump high‐volume flow cell design is simple to build and ideal for organisms that do not require feeding (i.e., eggs) and (b) a more advanced low‐volume double pump flow cell design that permits feeding, maintains optimal water height for water suppression, improves locking and shimming, and uses only a small recirculating volume, thus reducing the amount of xenobiotic required for testing. In addition, key experimental aspects including isotopic enrichment, water suppression, and 2D experiments for both 13C enriched and natural abundance organisms are discussed.

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“…Amino-acid selective SI labeling (AASIL) helps to discriminate the amino-acid type of each signal, independently of the triple resonance experiment-based sequential assignment. Therefore, it is especially useful for the signal assignment of difficult targets, such as large complex systems (Bertelsen et al 2009), low-solubility proteins (Cervantes et al 2013), and proteins in living cells (Hembram et al 2013). The dual selective labeling method, which utilizes both amide nitrogen and carbonyl carbon labeling, narrows down the assignment possibilities even further (Kainosho and Tsuji 1982), and as a consequence leads to the assignment, without the need for triple resonance experiments, of amino-acid pairs occurring only once in the sequence.…”

Section: Introductionmentioning

confidence: 99%

Stable isotope labeling strategy based on coding theory

et al. 2015

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We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as ''encoding and decoding'' processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells.

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An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells

Cited by 27 publications

References 41 publications

High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

Flow‐based in vivo NMR spectroscopy of small aquatic organisms

Stable isotope labeling strategy based on coding theory

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