A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

Autor: Miao Q; Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands., Zurlo E; Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, PO box 9504, 2300, RA, Leiden, The Netherlands., de Bruin D; Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, PO box 9504, 2300, RA, Leiden, The Netherlands., Wondergem JAJ; Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, PO box 9504, 2300, RA, Leiden, The Netherlands., Timmer M; Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands., Blok A; Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands., Heinrich D; Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, PO box 9504, 2300, RA, Leiden, The Netherlands.; Fraunhofer Institute for Silicate Research ISC, 97082, Würzburg, Germany., Overhand M; Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands., Huber M; Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, PO box 9504, 2300, RA, Leiden, The Netherlands., Ubbink M; Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands.
Jazyk: angličtina
Zdroj: Chemistry (Weinheim an der Bergstrasse, Germany) [Chemistry] 2020 Dec 18; Vol. 26 (71), pp. 17128-17133. Date of Electronic Publication: 2020 Nov 17.
DOI: 10.1002/chem.202002743
Abstrakt: 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.
(© 2020 The Authors. Published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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