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.