Probing Conformational Exchange in Weakly Interacting, Slowly Exchanging Protein Systems via Off-Resonance R 1ρ Experiments: Application to Studies of Protein Phase Separation

R 1ρ relaxation dispersion experiments are increasingly used in studies of protein dynamics on the micro- to millisecond time scale. Traditional R 1ρ relaxation dispersion approaches are typically predicated on changes in chemical shifts between corresponding probe spins, Δω GE, in the interconverting states. Here, we present a new application of off-resonance 15N R 1ρ relaxation dispersion that enables the quantification of slow exchange processes even in the limit where Δω GE = 0 so long as the spins in the exchanging states have different intrinsic transverse relaxation rates (ΔR 2 = R 2,E – R 2,G ≠ 0). In this limit, the dispersion profiles become inverted relative to those measured in the case where Δω GE ≠ 0, ΔR 2 = 0. The theoretical background to understand this effect is presented, along with a simplified exchange matrix that is valid in the limits that are germane here. An application to the study of the dynamics of the germ granule protein Ddx4 in a highly concentrated phase-separated state is described. Notably, exchange-based dispersion profiles can be obtained despite the fact that Δω GE ≈ 0 and ΔR 2 is small, ∼20–30 s–1. Our results are consistent with the formation of a significantly populated excited conformational state that displays increased contacts between adjacent protein molecules relative to the major conformer in solution, leading to a decrease in overall motion of the protein backbone. A complete set of exchange parameters is obtained from analysis of a single set of 15N off-resonance R 1ρ measurements recorded at a single static magnetic field and with a single spin-lock radio frequency field strength. This new approach holds promise for studies of weakly interacting systems, especially those involving intrinsically disordered proteins that form phase-separated organelles, where little change to chemical shifts between interconverting states would be expected, but where finite ΔR 2 values are observed.