Cleavage-resistant Protein Labeling with Hydrophilic Trityl Enables Distance Measurements In-Cell
| Autor: | Teresa D. Gluth, Benoit Driesschaert, Sunil Saxena, Kevin Singewald, Zikri Hasanbasri |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Materials science
Xenopus 010402 general chemistry Protein labeling Cleavage (embryo) 01 natural sciences Article chemistry.chemical_compound 0103 physical sciences Materials Chemistry Physical and Theoretical Chemistry Maleimide Longitudinal Relaxation Time 010304 chemical physics biology Electron Spin Resonance Spectroscopy Temperature Resonance Proteins biology.organism_classification 0104 chemical sciences Surfaces Coatings and Films Crystallography Deuterium chemistry Spin Labels Linker Hydrophobic and Hydrophilic Interactions |
| Zdroj: | J Phys Chem B |
| Popis: | 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 long-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. |
| Databáze: | OpenAIRE |
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