Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteinsin solution

A general method is presented for magnetic field alignment of proteins in s olution. By tagging a target protein with calmodulin saturated with paramag netic lanthanide ions it is possible to measure substantial residual dipola r couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate red uctase in this case. The engineered protein binds tightly to calmodulin sat urated with terbium, a paramagnetic lanthanide ion. By using only a short l inker region between the M13 and the target protein, some of the magnetic f ield alignment induced in the CaM(Tb3+)(4) is effectively transmitted to th e target protein (DHFR). H-1-N-15 HSQC IPAP experiments on the tagged compl ex containing N-15-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)(4) al low one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of H-1-N-15 HSQC spectra of N-15-DHFR-M13 alone and its complexes with CaM(Ca2+)(4) an d CaM(Tb3+)(4) indicated that (i) the structure of the target protein is no t affected by the complex formation and (ii) the spectra of the target prot ein are not seriously perturbed by pseudocontact shifts. The use of a relat ively large tagging group (CaM) allows us to use a lanthanide ion with a ve ry high magnetic susceptibility anisotropy (such as Tb3+) to give large ali gnments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).