Deconvoluting Monomer- and Dimer-Specific Distance Distributions between Spin Labels in a Monomer/Dimer Mixture Using...

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g., monomer and dimer), the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here, we propose incorporating an EPR longitudinal electron relaxation (T₁) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent T₁ between dipolar coupled electron spins measured from the inversion recovery time (τ_inv) dependence of the peak intensities in the T₁-edited DEER-derived probability P(r) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the T₁-edited DEER echo curves, recorded over a range of τ_inv values, permits the deconvolution of distances between spin labels originating from monomeric (longer T₁) and dimeric (shorter T₁) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex P(r) distance distributions. The utility of this approach is demonstrated for two systems, the β₁ adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.