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 (Tm). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the Tm 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 (T1) 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 Tm 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.