ARTICLE A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy Mathias A. S. Hass • Wei-Min Liu • Roman V. Agafonov • Renee Otten • Lien A. Phung • Jesika T. Schilder • Dorothee Kern • Marcellus Ubbink Received: 30 October 2014 / Accepted: 22 December 2014 / Published online: 8 January 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by charac- terizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxa- tion dispersion is the increase in the amplitude of disper- sion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dis- persion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements. Keywords Relaxation dispersion  Lanthanide binding tags  Protein dynamics  Paramagnetic NMR  Caged lanthanide NMR probe  Adenylate kinase Introduction The structure and dynamics of proteins form the basis of biomolecular function. Traditionally, structure and dynamics have been studied separately, presumably for practical experimental reasons: To determine a structure and simultaneously see it move is challenging indeed, although good progress has been made in considering nanosecond dynamics (Torda et al. 1990; Cornilescu et al. 1998; Bouvignies et al. 2006; Markwick et al. 2009). A major step forward in characterizing dynamics has been the development of new NMR methods, in particular relaxa- tion dispersion techniques (Palmer et al. 2001). These techniques have provided experimental support for the idea that the ground state of proteins coexists in a dynamic equilibrium with other conformations, whose energies are only slightly higher than that of the ground state. Several studies have suggested that these alternative conformations are likely to be functional, e.g. in enzyme catalysis (Vall- urupalli et al. 2007; Wang et al. 2007; Vallurupalli et al. 2008a, b; Bouvignies et al. 2011). This idea echoes the well-known concept of allostery, most prominently fea- tured in the Monod–Wyman–Changeux model (Monod et al. 1965). Experimental verification of such models is Electronic supplementary material The online version of this article (doi:10.1007/s10858-014-9894-3) contains supplementary material, which is available to authorized users. M. A. S. Hass  W.-M. Liu  J. T. Schilder  M. Ubbink (&) Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands e-mail: m.ubbink@chem.leidenuniv.nl R. V. Agafonov  R. Otten  L. A. Phung  D. Kern Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South Street, Waltham, MA 02454, USA 123 J Biomol NMR (2015) 61:123–136 DOI 10.1007/s10858-014-9894-3