A Gel‐Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells

Kubo, Shugo; Nishida, N.; Udagawa, Yuko; Takarada, Osamu; Ogino, Shuichi; Shimada, Ichio

doi:10.1002/anie.201207243

Cited by 95 publications

(119 citation statements)

References 16 publications

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“…by employing other selective isotope labeling techniques35 and the state-of-art reconstruction algorithms previously described, and suppressing cell death in the sample tube, thereby allowing NMR measurement times beyond 6 hours. The cell death suppression can be achieved using recent technology such as the Bioreactor system that continuously supplies fresh medium from outside the spectrometer36.…”

Section: Discussionmentioning

confidence: 99%

Improved in-cell structure determination of proteins at near-physiological concentration

Ikeya

Hanashima

Hosoya

et al. 2016

Sci Rep

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Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.

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Section: Discussionmentioning

confidence: 99%

Improved in-cell structure determination of proteins at near-physiological concentration

Ikeya

Hanashima

Hosoya

et al. 2016

Sci Rep

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“…This is accomplished by adding cell protectants, such as glycerol or sucrose, or by encapsulating the cells in alginate or agarose . Bioreactors, which sustain cells in the NMR spectrometer, have also been used to extend the time for data collection …”

Section: Introductionmentioning

confidence: 99%

“…Previous in vitro work indicated that α‐Syn engages in long‐range interactions between N‐ and C‐terminal residues, generating structures more compact than that of an extended polypeptide chain and occluding the NAC region. Paramagnetic relaxation enhancement (PRE) and electron paramagnetic resonance (EPR), double electron‐electron resonance (DEER) spectra were collected for A2780 and SK‐N‐SH cells that were electroporated with isotope‐enriched, maleimide‐DOTA‐tagged, and Gd(III)‐loaded α‐Syn. The results were similar to those obtained for purified α‐Syn, consistent with intramolecular interactions.…”

Section: Introductionmentioning

confidence: 99%

In‐Cell NMR Spectroscopy of Intrinsically Disordered Proteins

2019

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This review summarizes the results of in‐cell Nuclear Magnetic Resonance, NMR, spectroscopic investigations of the eukaryotic and prokaryotic intrinsically disordered proteins, IDPs: α‐synuclein, prokaryotic ubiquitin‐like protein, Pup, tubulin‐related neuronal protein, Tau, phenylalanyl‐glycyl‐repeat‐rich nucleoporins, FG Nups, and the negative regulator of flagellin synthesis, FlgM. The results show that the cellular behavior of IDPs may differ significantly from that observed in the test tube.

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“…Theconcentration of GB1 in the sf9 cells was predicted to approximately 129 mm (Supporting Information, Figure S1). [11] This strategy prolonged the lifetime of the cells in the NMR tube to be almost comparable to that under "optimum" condition in culture flasks,m aintaining > 90 % cell viability as well as protein stability in the cells for at least 24 hours (Supporting Information, Figures S2-4). In our previous work, approximately 80 %ofthe backbone NMR resonances of GB1 were assigned exclusively from 3D triple-resonance NMR spectra in sf9 cells, [5a] while 3D spectra for side-chain resonance assignment and 3D nuclear Overhauser effect spectroscopy (NOESY) spectra suffered from the short lifetime of the cells.…”

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confidence: 99%

“…Therefore,w ei ntroduced ab ioreactor system that supplies fresh medium into the NMR tube continuously. [11] This strategy prolonged the lifetime of the cells in the NMR tube to be almost comparable to that under "optimum" condition in culture flasks,m aintaining > 90 % cell viability as well as protein stability in the cells for at least 24 hours (Supporting Information, Figures S2-4). Moreover, the bioreactor is also effective for removing extracellular proteins (Supporting Information, Figure S3), thus guaranteeing that only proteins inside sf9 cells contribute to the incell NMR spectra.…”

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confidence: 99%

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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A Gel‐Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells

Cited by 95 publications

References 16 publications

Improved in-cell structure determination of proteins at near-physiological concentration

Improved in-cell structure determination of proteins at near-physiological concentration

In‐Cell NMR Spectroscopy of Intrinsically Disordered Proteins

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

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