Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins

Christoph Gmeiner (1) , Daniel Klose (1) , Elisabetta Mileo (2) , Valérie Belle (2) , Sylvain R.A. Marque (3) , Georg Dorn (1) , Frédéric H. T. Allain (1) , Bruno Guigliarelli (2) , Gunnar Jeschke (1) , Maxim Yulikov (1)

1 ETH Zürich - Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich]
2 BIP - Bioénergétique et Ingénierie des Protéines
3 ICR - Institut de Chimie Radicalaire

Abstract Image Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

Domains: Life Sciences [q-bio]; Chemical Sciences; Physics [physics]

Christoph Gmeiner, Daniel Klose, Elisabetta Mileo, Valérie Belle, Sylvain R.A. Marque, et al.. Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins. Journal of Physical Chemistry Letters, In press, pp.4852 - 4857. ⟨10.1021/acs.jpclett.7b02220⟩. ⟨hal-01596569⟩