In-cell NMR spectroscopy

Maldonado, Andres Y.; Burz, David S.; Shekhtman, Alexander

doi:10.1016/j.pnmrs.2010.11.002

Cited by 60 publications

(55 citation statements)

References 114 publications

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“…The advent of in-cell NMR provides means for direct investigation of macromolecules in living cells [191][192][193]. This method has been progressively applied to protein studies including structure determinations and protein-protein/ligand interactions [193][194][195][196].…”

Section: G-quadruplexes In Vivomentioning

confidence: 99%

NMR spectroscopy of G-quadruplexes

Adrian

Heddi

Phan

2012

Methods

283

245

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Section: G-quadruplexes In Vivomentioning

confidence: 99%

NMR spectroscopy of G-quadruplexes

Adrian

Heddi

Phan

2012

Methods

283

245

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“…It is therefore a powerful method for acquiring structural and functional information about biomolecules at atomic resolution in their natural, physiological environment [1][2][3][4][5] . In-cell NMR has been used successfully on bacterial cells to analyze protein structure 6 , dynamics 7,8 and interactions [9][10][11] .…”

Section: Introductionmentioning

confidence: 99%

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

2016

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In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

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“…I n-cell NMR represents a unique tool for characterizing physiological processes and their alteration in the 'natural' environment, that is, within a live cellular context, with an atomic-level approach [1][2][3][4][5][6][7][8][9] . This level of characterization is necessary to understand the molecular cause of cell malfunction in pathological states and to develop treatments and therapeutic protocols.…”

mentioning

confidence: 99%

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

et al. 2014

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Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

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In-cell NMR spectroscopy

Cited by 60 publications

References 114 publications

NMR spectroscopy of G-quadruplexes

NMR spectroscopy of G-quadruplexes

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

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