Long‐Range‐Distance NMR Effects in a Protein Labeled with a Lanthanide–DOTA Chelate

A two‐thiol reactive lanthanide–DOTA (1,4,7,10‐tetraazacyclododecane‐ N , N′ , N′′ , N′′′ ‐tetraacetic acid) chelate, CLaNP‐3 (CLaNP = caged lanthanide NMR probe), was synthesized for the rigid attachment to cysteine groups on a protein surface, and used to obtain long‐range‐distance information from the { 15 N, 1 H} HSQC spectra of the protein–lanthanide complex. The DOTA ring exhibits several isomers that are in exchange; however, single resonances were observed for most amide groups in the protein, allowing determination of a single, apparent magnetic‐susceptibility tensor. Pseudocontact shifts caused by Yb‐containing CLaNP‐3 were observed for atoms at 15–35 Å from the metal. By using Gd‐containing CLaNP‐3, relaxation effects were observed, allowing distances up to 30 Å from the paramagnetic center to be determined accurately. Similar results were obtained with a Gd–DTPA (diethylene‐triaminepentaacetic acid) chelate, CLaNP‐1, bound in the same bidentate manner to the protein. This study demonstrates that bidentate attachment of a paramagnetic probe enables determination of long‐range distances.