Efficient Orthogonal Spin Labeling of Proteins via Aldehyde Cyclization for Pulsed Dipolar EPR Distance Measurements

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