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 paramag netic Tb in C2+/calmodulin systems in solution allows the measurement of in terdomain NMR pseudocontact shifts and leads to magnetic alignment of the m olecule such that significant residual dipolar couplings can be measured. B oth these parameters can be used to provide structural information, Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domai n) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters, The nuclei in the C-domain experience the loca l magnetic field induced by the paramagnetic Tb ions bound to the other dom ain at distances of over 40 Angstrom from the Tb3+ ion, shifting the resona nces for these nuclei, In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the mo lecule so that a certain degree of alignment is produced due to interaction with the external magnetic field, In this way, dipolar couplings between n uclear spins are not averaged to zero due to solution molecular tumbling an d 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 dom ain with respect to the N-domain containing the Tb3+ ions. Pseudocontact sh ifts 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 h ave orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains h ave fixed orientations with respect to each other. The simultaneous measure ments of these parameters for systems with domains in fixed orientations sh ow great potential for the determination of the relative orientation of the domains. (C) 1999 Federation of European Biochemical Societies.