Site-directed spin labeling of proteins for distance measurements in vitro and in cells

Roser, Patrick; Schmidt, Moritz J.; Drescher, Malte; Summerer, Daniel

doi:10.1039/c6ob00473c

Cited by 89 publications

(82 citation statements)

References 93 publications

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“…For spin-labeled studies based on peptide synthesis, the nitroxide derivatized unnatural amino acid 1,1,6,6, - tetramethyl- N -oxyl-4-amino-4-carboxylic acid (TOAC) is a useful way to incorporate a nitroxide center [102]. In vivo incorporation of nitroxides using unnatural amino acid technology based on amber codon suppression has also been reported, both in Xenopus oocytes [103] and in E. coli [104,105]. Unnatural amino acid technologies can also enable two step spin labeling by incorporating amino acids with bioorthogonal reactive side chains, with the nitroxide subsequently coupled via click chemistry [106] [107].…”

Section: Spin Tagging In Magnetic Resonancementioning

confidence: 99%

“…EPR signals from inside the cell were invisible, while those on the exterior of the cell were detected, suggesting reduction upon transport into the cell. Similarly, in cells expressing proteins with the unnatural amino acid SLK-1, reduction of the EPR probe in cells and in lysate was reported, and prevented successful measurements in vivo [104,105,139]. It has been shown that 5-membered piperidine rings have improved stability in biological systems in comparison with the six membered pyrollidines typically used in DNP applications [140,141].…”

Section: Dnp In Cell Applications: Challenges and Opportunitiesmentioning

confidence: 99%

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New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Rogawski

McDermott

2017

Archives of Biochemistry and Biophysics

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Magic angle spinning solid state NMR studies of biological macromolecules [1–3] have enabled exciting studies of membrane proteins [4,5], amyloid fibrils [6], viruses, and large macromolecular assemblies [7]. Dynamic nuclear polarization (DNP) provides a means to enhance detection sensitivity for NMR, particularly for solid state NMR, with many recent biological applications and considerable contemporary efforts towards elaboration and optimization of the DNP experiment. This review explores precedents and innovations in biological DNP experiments, especially highlighting novel chemical biology approaches to introduce the radicals that serve as a source of polarization in DNP experiments.

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Section: Spin Tagging In Magnetic Resonancementioning

confidence: 99%

Section: Dnp In Cell Applications: Challenges and Opportunitiesmentioning

confidence: 99%

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Rogawski

McDermott

2017

Archives of Biochemistry and Biophysics

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“…To make progress in this field of research, it is necessary to understand the contributions to the loss of signal. One potential source of signal loss is cleavage of the disulfide bond of the typical methanothiosulfonate nitroxide side chain (called R1) in the cellular environment . Cleavage of the R1 side chain results in decreased protein labeling without a loss in overall signal, and therefore an increase in background noise, hindering the DEER experiment.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Nitroxide‐Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy

et al. 2017

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Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

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“…For this purpose several paramagnetic species can be chosen e.g., radical centers, metal ions, Cu 2+ or FeS clusters or nitroxide spin labels [7,8]. The choice of the spin label can be critical as it should be short enough to reduce its intrinsic flexibility but long enough to allow the native folding of the spin-labeled protein.…”

Section: Site-directed Spin Labelingmentioning

confidence: 99%

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

Wunnicke

Hänelt

2017

Crystals

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Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.

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Site-directed spin labeling of proteins for distance measurements in vitro and in cells

Cited by 89 publications

References 93 publications

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

Analysis of Nitroxide‐Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

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