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

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

Increasing the Modulation Depth of Gd^III-Based Pulsed Dipolar EPR Spectroscopy (PDS) with Porphyrin–Gd^III Laser-Induced Magnetic Dipole Spectroscopy

Distance determination with pulsed EPR has become an important technique for the structural investigation of biomacromolecules, with double electron–electron resonance spectroscopy (DEER) as the most important method. GdIII-based spin labels are one of the most frequently used spin labels for DEER owing to their stability against reduction, high magnetic moment, and absence of orientation selection. A disadvantage of GdIII–GdIII DEER is the low modulation depth due to the broad EPR spectrum of GdIII. Here, we introduce laser-induced magnetic dipole spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique. We show that the excited state of the porphyrin is not disturbed by the presence of the GdIII complex and that herewith modulation depths of almost 40% are possible. This is significantly higher than the value of 7.2% that was achieved with GdIII–GdIII DEER