Genetically Encoded Spin Labels for In Vitro and In-Cell EPR Studies of Native Proteins

Schmidt, Moritz J.; Fedoseev, Artem; Summerer, Daniel; Drescher, Malte

doi:10.1016/bs.mie.2015.05.023

Cited by 23 publications

(18 citation statements)

References 57 publications

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“…This result went along with low modulation depths in DEER measurements for doubly labeled thioredoxin, and no in-cell DEER experiment was reported. Nevertheless, an experimental comparison of 6 and the MTSL-derived label R1 by DEER revealed similar widths of the distance distributions, indicating that this lysine-derived ncAA is a useful probe for DEER measurements [55,56]. Moreover, rotamer libraries were established, enabling simulations of distance distributions obtained with 6 (Figure 6b,c).…”

Section: Direct Encoding Of Spin Labeled Noncanonical Amino Acidsmentioning

confidence: 87%

Expanding the Genetic Code for Site-Directed Spin-Labeling

Braun

Drescher

Summerer

2019

IJMS

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Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL–EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL–EPR studies.

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Section: Direct Encoding Of Spin Labeled Noncanonical Amino Acidsmentioning

confidence: 87%

Expanding the Genetic Code for Site-Directed Spin-Labeling

Braun

Drescher

Summerer

2019

IJMS

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“…. Surprisingly, it was reported that an unnatural amino acid carrying a five‐membered nitroxide can partially survive E. coli cell conditions. Wee will address later under which experimental conditions the latter conclusion was reached.…”

Section: Introductionmentioning

confidence: 92%

“…For in‐cell EPR‐experiments on spin‐labeled proteins, two methods have been explored: i) The expression of proteins with unnatural amino acids either containing an intrinsic stable paramagnetic center or enabling specific reactions with spin labels in living cells; ii) The insertion of a spin‐labeled recombinantly‐produced protein into cells via hypotonic swelling, electroporation, or micro‐injection into oocytes …”

Section: Introductionmentioning

confidence: 99%

“…Therefore, they are not suitable for in‐cell EPR measurements, unless strategical “tricks” are used, such as working with cell strains with less reducing agents, spin labeling outer membrane proteins, incubating spin‐labeled proteins with isolated organelles, etc. . Surprisingly, it was reported that an unnatural amino acid carrying a five‐membered nitroxide can partially survive E. coli cell conditions.…”

Section: Introductionmentioning

confidence: 99%

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gem‐Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility

et al. 2019

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The availability of bioresistant spin labels is crucial for the optimization of site‐directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin‐labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide‐ and maleimide‐functionalized spin labels based on the gem ‐diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem ‐dimethyl analogs by site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem ‐diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.

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“…4) has been genetically encoded using an evolved Methanosarcina mazei pyrrolysyl-tRNA-synthetase mutant, 79 allowing for the cotranslational incorporation of nitroxides directly in E. coli cells. 59,61 SLK-1 was introduced into multiple sites of GFP and thioredoxin and exhibited similar flexibility as MTSSL in simulations and in DEER distance measurements. 60…”

Section: Nitroxide Spin Labelsmentioning

confidence: 99%

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

et al. 2016

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Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells.

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Genetically Encoded Spin Labels for In Vitro and In-Cell EPR Studies of Native Proteins

Cited by 23 publications

References 57 publications

Expanding the Genetic Code for Site-Directed Spin-Labeling

Expanding the Genetic Code for Site-Directed Spin-Labeling

gem‐Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility

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

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