Membrane proteins, magic-angle spinning, and in-cell NMR

Pielak, Gary J.; Tian, Fang

doi:10.1073/pnas.1201502109

Cited by 8 publications

(7 citation statements)

References 22 publications

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“…In comparison with in-cell NMR 4,5 , in-cell EPR has two major advantages: low concentrations of spin labels can be used because EPR sensitivity per spin is much higher than that of NMR, and diamagnetic molecules in the vicinity of the spin label will not reduce the detection sensitivity of the signal, as EPR detects only unpaired electron spins. The latter advantage is particularly notable as, in contrast to structural studies conducted on isolated macromolecules, in in-cell experiments the macromolecules under investigation are often found in close proximity to many different cellular components, which could otherwise be associated with high background signal and decreased detection sensitivity of the desired signals.…”

Section: Comparison With Other Methodsmentioning

confidence: 99%

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

et al. 2012

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Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ~20 d.

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Section: Comparison With Other Methodsmentioning

confidence: 99%

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

et al. 2012

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“…As the cells used in in-cell NMR are alive, intact and contain complete cellular compartments, the obtained information is therefore very useful in biology, as well as other fields, such as drug discovery. Although structural studies of membrane proteins in living cells are of great interest for in-cell NMR, this review will mainly focus on in-cell NMR studies of water-soluble proteins carried out using solution-state NMR [61,65].…”

Section: In-cell Nmrmentioning

confidence: 99%

Applications of In-Cell NMR in Structural Biology and Drug Discovery

Kang

2019

IJMS

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In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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“…On the positive side, bacterial in-cell NMR samples are prepared quickly (90), and, provided that isotope-labeled proteins tumble freely in the cytoplasm and do not overly interact with cellular components such as membranes, in-cell NMR detection by solution-state methods is straightforward. Whenever target proteins are inserted into membranes, solid-state in-or on-cell NMR approaches are more suitable and are usually chosen (74). On the negative side, recombinant protein production is difficult to control, and absolute intracellular protein concentrations are hard to quantify and reproduce precisely.…”

Section: In-cell Nmr In Prokaryotesmentioning

confidence: 99%

“…In many instances, however, researchers wish to study large macromolecular assemblies or membrane proteins, which necessitate different approaches. High-resolution magic angle spinning (HR-MAS) solid-state NMR methods on cellular samples have been successfully employed to this end (74,78). Introduced in 2001 to characterize the abundant osmoregulated periplasmic glucans in Ralstonia solanacearum (103), HR-MAS methods have since been used to study protein structures in bacterial inclusion bodies (24,102), intracellular protein assemblies (77), membrane proteins in isolated membranes (32,46,48,58,79,80), and whole cells (47,80,99,105).…”

Section: Solid-state In-cell Nmrmentioning

confidence: 99%

Live Cell NMR

Freedberg

Selenko²

2014

Annu. Rev. Biophys.

134

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Ever since scientists realized that cells are the basic building blocks of all life, they have been developing tools to look inside them to reveal the architectures and mechanisms that define their biological functions. Whereas "looking into cells" is typically said in reference to optical microscopy, high-resolution in-cell and on-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful method that offers exciting new possibilities for structural and functional studies in and on live cells. In contrast to conventional imaging techniques, in- and on-cell NMR methods do not provide spatial information on cellular biomolecules. Instead, they enable atomic-resolution insights into the native cell states of proteins, nucleic acids, glycans, and lipids. Here we review recent advances and developments in both fields and discuss emerging concepts that have been delineated with these methods.

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Membrane proteins, magic-angle spinning, and in-cell NMR

Cited by 8 publications

References 22 publications

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Applications of In-Cell NMR in Structural Biology and Drug Discovery

Live Cell NMR

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