Open AccessCleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell
Author(s) -
Zikri Hasanbasri,
Kevin Singewald,
Teresa D. Gluth,
Benoît Driesschaert,
Sunil Saxena
Publication year - 2021
Publication title -
the journal of physical chemistry. b
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 392
eISSN - 1520-6106
pISSN - 1520-5207
DOI - 10.1021/acs.jpcb.1c02371
Subject(s) - cleavage (geology) , maleimide , linker , xenopus , chemistry , relaxation (psychology) , electron paramagnetic resonance , analytical chemistry (journal) , materials science , biophysics , nuclear magnetic resonance , chromatography , polymer chemistry , physics , psychology , social psychology , biochemistry , fracture (geology) , biology , computer science , composite material , gene , operating system
Sensitive in-cell distance measurements in proteins using pulsed-electron spin resonance (ESR) require reduction-resistant and cleavage-resistant spin labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time ( T m ). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the T m of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d 24 ). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells . Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time ( T 1 ) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T m of mOX063-d 24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d 24 provides highly sensitive distance measurements both in vitro and in-cells .