DEER distance measurements on trityl/trityl and Gd(iii)/trityl labelled proteins

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.