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
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- 作者：Elisabetta Mileo ; Emilien Etienne ; Marl猫ne Martinho ; R茅gine Lebrun ; Val茅rie Roubaud ; Paul Tordo ; Brigitte Gontero ; Bruno Guigliarelli ; Sylvain R. A. Marque ; Val茅rie Belle
- 刊名：Bioconjugate Chemistry
- 出版年：2013
- 出版时间：June 19, 2013
- 年：2013
- 卷：24
- 期：6
- 页码：1110-1117
- 全文大小：349K
- 年卷期：v.24,no.6(June 19, 2013)
- ISSN：1520-4812
文摘
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.