Probing Conformational Exchange in Weakly Interacting, Slowly Exchanging Protein Systems via Off-Resonance R_1ρ...

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 (ΔR₂ = 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, ΔR₂ = 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 ΔR₂ 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 ΔR₂ values are observed.