A Two‐Armed Probe for In‐Cell DEER Measurements on Proteins. Issue 71 (17th November 2020)

• Record Type: Journal Article
• Authors: Miao, Qing; Zurlo, Enrico; de Bruin, Donny; Wondergem, Joeri A. J.; Timmer, Monika; Blok, Anneloes; Heinrich, Doris; Overhand, Mark; Huber, Martina; Ubbink, Marcellus
• Is Part Of: Chemistry. Volume 26:Issue 71(2020)
• Page Start: 17128
• Page End: 17133
• Publication Date: 2020-11-17
• Subjects: double electron–electron resonance (DEER) -- EPR spectroscopy -- gadolinium -- protein structures -- spin labels; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.202002743
• Languages: English
• ISSNs: 0947-6539

Abstract

Abstract: 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. Abstract : Two armed and ready ! A two armed, thus rigid, Gd III ‐CLaNP13 probe for double electron–electron resonance (DEER) measurements on proteins can be used to measure nanometer distances in cells with high precision.