Paramagnetic relaxation enhancements for the characterization of the conformational heterogeneity in two-domain proteins

Multidomain proteins are often composed of rigid domains that can reorient in solution more or less freely. Calmodulin (CaM) is a two domain protein which can experience a large degree of conformational freedom thanks to a mobile linker connecting the N-terminal and C-terminal domains of the protein. The maximum occurrences (MOs) of the possible protein conformations have been analyzed using the paramagnetic relaxation enhancements (PREs) induced by a gadolinium( III ) ion together with the paramagnetic pseudocontact shift and residual dipolar coupling restraints measured in the presence of terbium( III ), thulium( III ) or dysprosium( III ) ions. The results suggest that the PREs provide complementary information useful for improving the description of the conformational heterogeneity of the protein. The data, acquired at 298 K and at 278 K, suggest that compact conformations are disfavoured by decreasing the temperature.