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 <i>Escherichia coli</i>, 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.