A Reactive, Rigid Gd^III Labeling Tag for In-Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μm.