Fig. 2. Experimental strategy for the attachment of lipid anchor and fluorophores to enable FRET or EPR measurements of membrane bound Ras proteins. In order to perform FRET or EPR measurements on membrane-bound Ras, two site specific protein modifications were necessary; one to attach the lipid anchor to the protein and another for the fluorophore or spin label. (A) The lipid anchor is attached via a maleimide group to ensure membrane binding. (B) The fluorophore is coupled site specifically via copper(i) catalyzed alkyne–azide cycloaddition to the previously incorporated unnatural amino acid (Fig. S2†). (C) A Ras dimer structural model with attached lipid anchors and the fluorophore pair T124-Atto 655/T124-Atto 532. (D) An overview of the labeling sites and the experimentally obtained FRET and EPR distances between two membrane bound full-length N-Ras-GDP proteins. The previously reported GDP distance was used.²¹.