Efficient Orthogonal\nSpin Labeling of Proteins via\nAldehyde Cyclization for Pulsed Dipolar EPR Distance Measurements

Pulsed dipolar electron paramagnetic resonance (PD-EPR)\nmeasurement\nis a powerful technique for characterizing the interactions and conformational\nchanges of biomolecules. The extraction of these distance restraints\nfrom PD-EPR experiments relies on manipulation of spin–spin\npairs. The orthogonal spin labeling approach offers unique advantages\nby providing multiple distances between different spin–spin\npairs. Here, we report an efficient orthogonal labeling approach based\non exploiting the cyclization between the 1,2-aminothiol moiety in\na protein (e.g., the N-terminal cysteine) with the aldehyde group\nin a spin label and a thiol substitution (or addition) reaction with\na different spin label. We demonstrated that this orthogonal spin\nlabeling method enables high accuracy and precision of multiple protein\ndistance constraints through the PD-EPR measurement from a single\nsample. This spin labeling approach was applied to characterize the\noligomeric state of the trigger factor (TF) protein of Escherichia coli, an important protein chaperone,\nin solution and cell lysates by distance measurements between different\nspin–spin pairs. Contrary to popular belief, TF exists mainly\nin the monomeric state and not as a dimer in the cell lysate.