A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies.
Ganesan KarthikeyanAlessio BonucciGilles CasanoGuillaume GerbaudSébastien AbelVirginie ThoméLaurent KodjabachianAxel MagalonBruno GuigliarelliValérie BelleOlivier OuariElisabetta MileoPublished in: Angewandte Chemie (International ed. in English) (2018)
Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing 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 nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. 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 is presented. 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.
Keyphrases
- single molecule
- induced apoptosis
- room temperature
- density functional theory
- cell cycle arrest
- single cell
- binding protein
- protein protein
- cell therapy
- amino acid
- case control
- endoplasmic reticulum stress
- high resolution
- signaling pathway
- mesenchymal stem cells
- stem cells
- risk assessment
- mass spectrometry
- hiv infected
- men who have sex with men
- bone marrow
- human immunodeficiency virus
- small molecule
- cell proliferation
- solid state