Conformationally locked lanthanide chelating tags for convenient pseudocontact shift protein nuclear magnetic resonance spectroscopy

Pseudocontact shifts (PCS) generated by lanthanide chelating tags yield valuable restraints for investigating protein structures, dynamics and interactions in solution. In this work, dysprosium-, thulium- and terbium-complexes of eight-fold methylated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tags [DOTA-M8-(4R4S)-SSPy] are presented that induce large pseudocontact shifts up to 5.5ppm and adopt exclusively the square antiprismatic conformation. This is in contrast to our earlier findings on complexes of the stereoisomeric DOTA-M8-(8S)-SSPy, where significant amounts of the twisted square antiprismatic conformer for the Dy tag were observed. The Dy-, Tm-, Tb- and Lu-complexes of DOTA-M8-(4R4S)-SSPy were conjugated to ubiquitin S57C and selectively N-15 leucine labeled human carbonic anhydrase II S50C, resulting in only one set of signals. Furthermore, we investigated the conformation of the thulium- and dysprosium-complexes in vacuo and with implicit water solvent using density functional theory calculations. The calculated energy differences between the two different conformations (7.0-50.5kJ/mol) and experimental evidence from the corresponding ytterbium- and yttrium-complexes clearly suggest a SAP [()] geometry for the complexes presented in this study. The lanthanide chelating tag studied in this work offer insights into the solution structure of proteins by inducing strong pseudocontact shifts, show different tensor properties compared to its predecessor, enables a convenient assignment procedure, is accessed by a more economic synthesis than its predecessor and constitutes a highly promising starting point for further developments of lanthanide chelating tags.