Diversification of EPR signatures in site directed spin labeling using a β-phosphorylated nitroxide

• DOI: 10.1039/c3cp54816c
• Publication Date: 2014-01-02T15:02:40Z
• Authors: Nolwenn Le Breton; Marlène Martinho; Kuanysh Kabytaev; Jérémie Topin; Elisabetta Mileo; David Blocquel; Johnny Habchi; Sonia Longhi; Antal Rockenbauer; Jérôme Golebiowski; Bruno Guigliarelli; Sylvain R. A. Marque; Valérie Belle
• Keywords: Site-directed spin labeling; Spin label; Folding (DSP implementation)

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading classical 3-line spectra whose spectral shapes indicative of the environment and thus constitute good reporters modifications. However, similarity these precludes probing two regions protein partner simultaneously. To overcome limitation due weak diversity label shapes, we designed new β-phosphorylated giving 6-line spectra. This paper describes synthesis this label, its grafting at four different positions model able undergo an induced α-helical folding characterization by spectroscopy. For comparative purposes, has been grafted same protein. The ability report was evaluated analyzing shape modifications either presence secondary structure stabilizer (trifluoroethanol) Taken together results demonstrate that phosphorylated gives distinguishable signature which from subtle larger transitions, as efficiently label. As complementary approach, molecular dynamics (MD) calculations were performed gain further insights into binding process between labeled NTAIL PXD. MD revealed does not disturb interaction reinforced conclusion probe local environments study represents important step forward extension panoply SDSL-EPR approaches.