Gd(III)-PyMTA Label Is Suitable for In-Cell EPR

Abstract

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd­(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of <i>Xenopus laevis</i> oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP₁₀CP₁₀CP₁₀-NH₂ was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into <i>Xenopus laevis</i> oocytes, and the Gd­(III)–Gd­(III) distances were determined by double electron–electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd­(III) ion and the C_α of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂ is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix