Inter-Domain Dynamics in a Two-Domain Protein Studied by NMR

Domain orientation and dynamics often play an important role in regulation of multidomain proteins function. Here we consider a two-domain system, Lys48-linked di-ubiquitin (Ub$_{2})$, which is the simplest model of the polyubiquitin chain involved in the ubiquitin-proteasome pathway. Under physiological conditions Ub$_{2}$ adopts a compact conformation, in which the functionally important hydrophobic residues are sequestered at the interface between the two Ub$_{2}$ domains. Here we present a dynamic model that combines the anisotropic overall rotational diffusion with intra- and interdomain dynamics. This model describes the interdomain motion as a transition between two distinct conformational states. The model is applied to experimental $^{15}$N relaxation data for Lys48-linked Ub$_{2}$ acquired at neutral (pH 6.8) and acidic (pH 4.5) conditions. The model provides complete picture of Ub$_{2}$ domain mobility including domain orientations, time scales of domain motions, and occupation probabilities for both states of Ub$_{2}$. The obtained results are consistent with independent data on chemical shift perturbation mapping and spin labeling.