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Title: Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell
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-d24). 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-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells.
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The Journal of Physical Chemistry B
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National Science Foundation
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