Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 26, Issue 71, Pages 17128-17133Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202002743
Keywords
double electron– electron resonance (DEER); EPR spectroscopy; gadolinium; protein structures; spin labels
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Funding
- Chinese Scholarship Council (CSC) [201506870013]
- Netherlands Organization for Scientific Research (NWO) [711.014.003]
- Fraunhofer Society for the Fraunhofer Attract grant 3DNanoCell
- Fraunhofer ICON grant BioSensing, NWO/OCW
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The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd-III complexes, Gd-III-CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly Gd-III labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.
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