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Issue 40, 2019
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Probing the dynamic properties of two sites simultaneously in a protein–protein interaction process: a SDSL-EPR study

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Abstract

During molecular processes, protein flexibility is a fundamental property allowing protein–protein interaction. Following structural changes during these interactions is then of crucial interest. Site-Directed Spin Labeling (SDSL) combined to EPR spectroscopy is a powerful technique to follow structural modifications within proteins and during protein–protein interactions. Usual nitroxide labels target cysteine residues and afford a 3-line spectrum, whose shape is informative of the structural environment of the label. However, it is not possible to probe two regions of a protein or two partner proteins at the same time because of the overlapping of EPR signatures. Previously, we reported the design and the characterization of a spin label based on a β-phosphorylated (PP) nitroxide yielding a 6-line spectrum. Here, we report the use of two labels with different EPR signatures, namely maleimido-proxyl (P) and PP, to follow structural changes during a protein–protein interaction process in one single experiment. As a model system, we chose a disordered protein that undergoes an induced α-helical folding upon binding to its partner. We show that the EPR spectrum of a mixture of labeled interacting proteins can be analyzed in terms of structural changes during the interaction. This study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.

Graphical abstract: Probing the dynamic properties of two sites simultaneously in a protein–protein interaction process: a SDSL-EPR study

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Publication details

The article was received on 22 Aug 2019, accepted on 26 Sep 2019 and first published on 27 Sep 2019


Article type: Paper
DOI: 10.1039/C9CP04660G
Phys. Chem. Chem. Phys., 2019,21, 22584-22588

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    Probing the dynamic properties of two sites simultaneously in a protein–protein interaction process: a SDSL-EPR study

    N. Le Breton, S. Longhi, A. Rockenbauer, B. Guigliarelli, S. R. A. Marque, V. Belle and M. Martinho, Phys. Chem. Chem. Phys., 2019, 21, 22584
    DOI: 10.1039/C9CP04660G

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