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

摘要: 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 protein via redox stable thioether bond. The performance of the was demonstrated W-band double electron-electron resonance (DEER) distance measurements doubly trityl-labelled mutants immunoglobulin G-binding 1 (GB1) ubiquitin. For both CT02MA-labelled measured, by applying chirped pump pulse(s), relatively narrow distributions, comparable those obtained with same labelled BrPy-DO3MA-Gd(iii). We noticed, however, that sample contained some free CT02MA difficult remove at purification step. Dual labelling ubiquitin one tag BrPy-DO3MA-Gd(iii) achieved as well trityl-Gd(iii) distribution facilitated use dual mode cavity combination pulse. also measured Gd(iii)-Gd(iii) this sample, showing procedure not fully selective. Nevertheless, these demonstrate potential high sensitivity Gd(iii)-trityl DEER proteins, can be further exploited designing orthogonal Gd(iii)/trityl schemes.