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2020
Journal Article
Title
A Two-Armed Probe for In-Cell DEER Measurements on Proteins
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 GdIII complexes, GdIII-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 GdIII 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.
Author(s)
Miao, Qing
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, Netherlands
Zurlo, Enrico
Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands
Bruin, Donny de
Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands
Wondergem, Joeri A.J.
Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands
Timmer, Monika
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, Netherlands
Blok, Anneloes J.
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, Netherlands
Overhand, Mark
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, Netherlands
Huber, Martina M.
Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands