In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag.

Double electron-electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS). Gd(III)-Gd(III) DEER measurements were carried out on cells frozen after different incubation times, following delivery to test the stability of the spin label inside the cell. For both delivery methods, it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd(III) and considering the presence of Mn(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd(III)/Mn(II) exchange with endogenous Mn(II). These experiments also showed that the relative Gd(III)/Mn(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.