An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub ). As expected, cyclization increased the thermal stability of Ub and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub . Furthermore, cyclization repressed the interdomain motion in Ub , but cyclic Ub still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub . This article is protected by copyright. All rights reserved.