Validation of a lanthanide tag for the analysis of protein dynamics by paramagnetic NMR spectroscopy

Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic (1)H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu(3+), Yb(3+), or Tm(3+) was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c. The paramagnetic center causes large relaxation dispersion effects for two attachment sites, suggesting that local dynamics of the protein at the attachment site causes mobility of the paramagnetic center. At one site the relaxation dispersions are small and limited to the immediate environment of the tag. It is concluded that paramagnetic relaxation dispersion could represent a sensitive method to probe protein dynamics. However, the selection of a rigid attachment site is of critical importance.