title: Enlarging the panoply of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR): sensitive and selective spin-labeling of tyrosine using an isoindoline-based nitroxide.
abstract: Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
authors: Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France.
authors: Elisabetta
authors: E
authors: Mileo
authors: Emilien
authors: E
authors: Etienne
authors: Marlène
authors: M
authors: Martinho
authors: Régine
authors: R
authors: Lebrun
authors: Valérie
authors: V
authors: Roubaud
authors: Paul
authors: P
authors: Tordo
authors: Brigitte
authors: B
authors: Gontero
authors: Bruno
authors: B
authors: Guigliarelli
authors: Sylvain R A
authors: SR
authors: Marque
authors: Valérie
authors: V
authors: Belle
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D054833:Isoindoles / Q000737:chemistry*; D015394:Molecular Structure; D009569:Nitric Oxide / Q000138:chemical synthesis / Q000737:chemistry*; D013113:Spin Labels; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D054833:Isoindoles; D013113:Spin Labels; D009569:Nitric Oxide; D014443:Tyrosine