title: Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution.
abstract: A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar 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 reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)4 is effectively transmitted to the target protein (DHFR). 1H-15N HSQC IPAP experiments on the tagged complex containing 15N-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)4 allow 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 1H-15N HSQC spectra of 15N-DHFR-M13 alone and its complexes with CaM(Ca2+)4 and CaM(Tb3+)4 indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).
authors.affiliation: Molecular Structure Division, National Institute for Medical Research, The Ridgeway, London, UK. jfeeney@nimr.mrc.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Feeny
authors.forename: B
authors.initials: B
authors.lastname: Birdsall
authors.forename: A F
authors.initials: AF
authors.lastname: Bradbury
authors.forename: R R
authors.initials: RR
authors.lastname: Biekofsky
authors.forename: P M
authors.initials: PM
authors.lastname: Bayley
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D007780:Lacticaseibacillus casei; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D046911:Macromolecular Substances; D008280:Magnetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry*; D013725:Terbium / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; D028581:Lanthanoid Series Elements; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012996:Solutions; D013725:Terbium; D013762:Tetrahydrofolate Dehydrogenase