Cysteine-Specific Cu2+ Chelating Tags Used as Paramagnetic Probes in Double Electron Electron Resonance

Double electron electron resonance (DEER) is an attractive technique that is utilized for gaining insight into protein structure and dynamics via nanometer-scale distance measurements. The most commonly used paramagnetic tag in these measurements is a nitroxide spin label, R1. Here, we present the application of two types of high-affinity Cu²⁺ chelating tags, based on the EDTA and cyclen metal-binding motifs as alternative X-band DEER probes, using the B1 immunoglobulin-binding domain of protein G (GB1) as a model system. Both types of tags have been incorporated into a variety of protein secondary structure environments and exhibit high spectral sensitivity. In particular, the cyclen-based tag displays distance distributions with comparable distribution widths and most probable distances within 1–3 A when compared to homologous R1 distributions. The results display the viability of the cyclen tag as an alternative to the R1 side chain for X-band DEER distance measurements in proteins.