New spin labeling approaches

Electron paramagnetic resonance (EPR) techniques, specifically double electron-electron resonance DEER (or pulsed electron double resonance, PELDOR) are highly effective for determining the distances between two strategic sites in biomolecules such as proteins, nucleic acids, and their assemblies. These are usually carried out in frozen solutions and provide, for example, sparse structural information that can be used for tracking conformation changes upon ligand/substrate binding. They can be used as constraints for modeling of unknown structures, and can elucidate how individual protein subunits with known structures assemble into larger structures. At the heart of this methodology lies the controlled labeling of the molecules of interest with paramagnetic probes, between which the distances are measured. These could be either intrinsic paramagnetic centers such as transition metal ions and radicals or artificially introduced spin labels.

The field of spin labeling has been dominated by nitroxide stable radicals since it was first introduced by McConnell in the 1960s. Although highly popular to date, at conventional EPR frequencies DEER sensitivity limitations leave many important problems outside its applicability range. In addition, nitroxides have limited stability within living cells.

The above-mentioned limitations prompted my lab, together with our collaborators, to introduce new spin labels for DEER measurements at Q- and W-band frequencies. These are based on Gd3+, which is a half-integer, high-spin ion (S=7/2) with half-filled valence f orbitals that exhibit high EPR sensitivity at high magnetic fields. As opposed to nitroxides, they are stable in the cell; this is most important for future development of in-cell DEER.

Since the introduction of Gd3+-Gd3+ DEER, other spin labels have been added to the DEER repertoire like trityl radicals, Cu(II), Mn(II) and new types of nitroxides that are expected to be biocompatible, concurrently to novel labeling schemes. The availability of several types of spin labels has led to PD (pulse dipolar)-EPR measurements on systems where the labels within the pair are chemically different and thus spectroscopically different. This approach has advantages in some cases and allows measuring more than one distance per sample and paves the way to new applications. Our lab continues to work in this direction and currently employs expansion of the genetic code for unnatural amino acid incorporation into proteins, as site-selective labeling for in-cell applications.