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

Abstract

This work shows that the partial replacement of diamagnetic Ca 2+ by paramagnetic Tb 3+ in Ca 2+ /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 Tb 3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca 2+ 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 3+ ions bound to the other domain at distances of over 40 A î from the Tb 3+ ion, shifting the resonances for these nuclei. In addition, the Tb 3+ 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 15 N-1 H 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 Tb 3+ ions. Pseudocontact shifts for NH groups and 1 H-15 N 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.z 1999 Federation of European Biochemical Societies.

NMR and molecular dynamics studies of the interaction of melatonin with calmodulin

“…As shown in previous work, the Tb 3+ added binds preferentially to the N-domain of CaM (Biekofsky et al 1999 ) 2 -calmodulin (where I and II indicate the binding sites of the N-domain, III and IV those of the Cdomain). Figure 5 shows the four peaks corresponding to C-domain residues Ile 100 and Val 136.…”

“…This allows the measurement of interdomain NMR pseudocontact shifts (PCS) and of residual dipolar couplings (RDC) induced by magnetic alignment (Biekofsky et al 1999).…”

“…The degree of alignment of the Tb 3+ -free C-domain depends on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb 3+ ions (Biekofsky et al 1999 (Biekofsky et al 1999). Table 2 presents data measured for selected residues in these three systems to provide examples of the range of values obtained.…”

Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

“…It has been also observed that both pcs and rdc were sizably smaller than predicted by assuming that the x-ray conformation was maintained in solution, consistent with the presence of multiple conformations. The presence of a mixture of species deriving from lanthanide binding to different calcium sites apparently prevented a deeper analysis (28).…”

Nuclear Magnetic Resonance of Membrane-Associated Peptides and Proteins

“…However, the measured values of the RDCs are of similar magnitude(−3 to +4 Hz) to those observed with paramagnetic metals tightly bound to single sites in calcium-binding proteins 16 and other metalloproteins. 17,18 Even though fd coat protein has little sequence similarity to Pf1 coat protein, its overall structural properties are the same. It, too, has an adventitious lanthanide-binding site, consisting of residues 2 through 9 (EGDDPAKA) ( Membrane Proteins with Engineered "EF-Hand"-Not all membrane proteins have adventitious lanthanide binding sites.…”