title: A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy.
abstract: NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing 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 relaxation dispersion is the increase in the amplitude of dispersion 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 dispersion 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.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Mathias A S
authors.initials: MA
authors.lastname: Hass
authors.forename: Wei-Min
authors.initials: WM
authors.lastname: Liu
authors.forename: Roman V
authors.initials: RV
authors.lastname: Agafonov
authors.forename: Renee
authors.initials: R
authors.lastname: Otten
authors.forename: Lien A
authors.initials: LA
authors.lastname: Phung
authors.forename: Jesika T
authors.initials: JT
authors.lastname: Schilder
authors.forename: Dorothee
authors.initials: D
authors.lastname: Kern
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D000263:Adenylate Kinase / Q000032:analysis / Q000737:chemistry*; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation
chemical_list: D028581:Lanthanoid Series Elements; D000263:Adenylate Kinase