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Khlebtsov, Nikolai G.; Kovler, L. A.; Загирова, С. В.; Khlebtsov, Boris N.; Богатырев, В. А.

doi:10.1023/a:1016718324830

Cited by 8 publications

(5 citation statements)

References 10 publications

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“…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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confidence: 82%

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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confidence: 82%

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

Thoma

Burmann

2020

Biochemistry

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“…Another key advantage of using this effective sphere model is that it enables the retrieval of vesicle loading even when the refractive index of the lipid is unknown. Indeed, there are few reports , of lipid refractive index, especially as a function of wavelength.…”

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confidence: 99%

Measuring Vesicle Loading with Holographic Microscopy and Bulk Light Scattering

Tran,

Lowe,

Deckel

et al. 2024

ACS Phys. Chem Au

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We report efforts to quantify the loading of cellsized lipid vesicles using in-line digital holographic microscopy. This method does not require fluorescent reporters, fluorescent tracers, or radioactive tracers. A single-color LED light source takes the place of conventional illumination to generate holograms rather than bright field images. By modeling the vesicle's scattering in a microscope with a Lorenz−Mie light scattering model and comparing the results to data holograms, we are able to measure the vesicle's refractive index and thus loading. Performing the same comparison for bulk light scattering measurements enables the retrieval of vesicle loading for nanoscale vesicles.

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“…This effect is due to the dependence of the total extinction cross-section of anisotropic aggregates on their orientation. The theory of orientational turbidimetric effect was generalised 84 by inclusion of the interaction between dipoles in the fractal aggregate. The magnitude of the effect was calculated for fractal clusters of different size grown under specific conditions and then evaluated experimentally.…”

Section: Sqmentioning

confidence: 99%

The characteristics of fractal disperse systems

Roldughin¹

2003

Russ. Chem. Rev.

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Experimental methods of determination of the fractal Experimental methods of determination of the fractal dimensions of fractal aggregates are described. The results of dimensions of fractal aggregates are described. The results of studies on aggregation of colloidal particles in different regimes studies on aggregation of colloidal particles in different regimes are discussed. Data on the hydrodynamics of colloid dispersions are discussed. Data on the hydrodynamics of colloid dispersions containing fractal aggregates and on the rheology of fractal gels containing fractal aggregates and on the rheology of fractal gels are presented. Capillary phenomena in fractal systems as well as are presented. Capillary phenomena in fractal systems as well as the structural and mechanical properties of aerogels and peculiar the structural and mechanical properties of aerogels and peculiar features of the optical properties of fractal aggregates are features of the optical properties of fractal aggregates are described. The bibliography includes 395 references described. The bibliography includes 395 references. .

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Untitled

Cited by 8 publications

References 10 publications

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

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

Measuring Vesicle Loading with Holographic Microscopy and Bulk Light Scattering

The characteristics of fractal disperse systems

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