A bioresistant nitroxide spin label for in-cell EPR spectroscopy: in vitro and in oocytes protein structural dynamics studies.
 

A bioresistant nitroxide spin label for in-cell EPR spectroscopy: in vitro and in oocytes protein structural dynamics studies.


Approaching proteins structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Due to its absolute sensitivity and to its selectivity to paramagnetic species, Site-Directed Spin Labeling (SDSL) combined with Electron Paramagnetic Resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxyde-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. In this work we present the design and synthesis of the first maleimido-proxyl-based spin label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.

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