Recording In-Cell NMR-Spectra in Living Mammalian Cells

Matečko-Burmann, Irena; Burmann, Björn M.

doi:10.1007/978-1-0716-0524-0_44

Cited by 4 publications

(2 citation statements)

References 23 publications

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“…Secondary chemical shift values of the sequence-specific assigned residues of purified CpxP agree well with those of available crystal structures , (Figure B and Figure S3), indicating a mostly α-helical structure of purified CpxP in solution and, consequently, inside of OMVs. To assess the protein concentration inside OMVs and to test whether it resembles the concentration in the natural periplasmic environment, we used an approach adapted from in-cell NMR spectroscopy. − We integrated δ 2 [ 1 H] one-dimensional cross sections from 2D 15 N– 1 H TROSY spectra of purified CpxP at different concentrations and compared the signal intensities determined with CpxP-enriched OMVs, resulting in a total CpxP concentration of ≈86 μM in the NMR sample (Figure S5A,B). We then estimated the total vesicle volume from the turbidity of the NMR sample by first determining the vesicle radius from the slope of logarithmized optical density spectra (Figure S5C,D) followed by determining the corresponding number concentration from the optical density. − This resulted in an estimated CpxP concentration of ∼1.7 mM inside OMVs and is in agreement with values determined for periplasmic α-synuclein in previous studies …”

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High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles

Thoma

Burmann

2020

Biochemistry

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The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from Escherichia coli as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from E. coli using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution in situ NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membraneattached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows in situ analysis of the structure and dynamics of proteins twice the size compared to the current in-cell NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.

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High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles

Thoma

Burmann

2020

Biochemistry

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“…Investigating the role of molecular chaperones by sophisticated in-cell NMR spectroscopy in living mammalian cells [34,56,57] enabled us to directly study the effect of chaperone inhibition in the cellular context. Inhibiting the two main molecular chaperones, Hsc70 and Hsp90, indicated that the release of α-synuclein leads to a direct interaction with cellular membranes, as evident from the characteristic interaction pattern for the first 100 residues [34,56,58].…”

Section: Chaperone: α-Synuclein Interplaymentioning

confidence: 99%

Keeping α-Synuclein at Bay: A More Active Role of Molecular Chaperones in Preventing Mitochondrial Interactions and Transition to Pathological States?

Aspholm

Matečko-Burmann

Burmann

2020

Life

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The property of molecular chaperones to dissolve protein aggregates of Parkinson-related α-synuclein has been known for some time. Recent findings point to an even more active role of molecular chaperones preventing the transformation of α-synuclein into pathological states subsequently leading to the formation of Lewy bodies, intracellular inclusions containing protein aggregates as well as broken organelles found in the brains of Parkinson’s patients. In parallel, a short motif around Tyr39 was identified as being crucial for the aggregation of α-synuclein. Interestingly, this region is also one of the main segments in contact with a diverse pool of molecular chaperones. Further, it could be shown that the inhibition of the chaperone:α-synuclein interaction leads to a binding of α-synuclein to mitochondria, which could also be shown to lead to mitochondrial membrane disruption as well as the possible proteolytic processing of α-synuclein by mitochondrial proteases. Here, we will review the current knowledge on the role of molecular chaperones in the regulation of physiological functions as well as the direct consequences of impairing these interactions—i.e., leading to enhanced mitochondrial interaction and consequential mitochondrial breakage, which might mark the initial stages of the structural transition of α-synuclein towards its pathological states.

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