NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb in C2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information, Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters, The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb ions bound to the other domain at distances of over 40 Angstrom from the Tb3+ ion, shifting the resonances for these nuclei, In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field, In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond N-15-H-1 splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and H-1-N-15 residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains. (C) 1999 Federation of European Biochemical Societies.