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Journal of Biomolecular NMR

, Volume 61, Issue 2, pp 123–136 | Cite as

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 UbbinkEmail author
Article

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.

Keywords

Relaxation dispersion Lanthanide binding tags Protein dynamics Paramagnetic NMR Caged lanthanide NMR probe Adenylate kinase 

Notes

Acknowledgments

Financial support was provided by the Netherlands Organisation for Scientific Research grants 700.10.407 (M.A.S.H) and 700.58.441 (M.U., W.M.L. and J.T.S.), the Howard Hughes Medical Institute and the Office of Basic Energy Sciences, Catalysis Science Program, U.S. Dept. of Energy, award DE-FG02-05ER15699 and National Institutes of Health, award GM100966-01 (R.V.A., L.A.P., R.O. and D.K.), and R.O. is a HHMI Fellow of the Damon Runyon Cancer Research Foundation, DRG-2114-12.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10858_2014_9894_MOESM1_ESM.pdf (3.9 mb)
Supplementary material 1 (PDF 4003 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Mathias A. S. Hass
    • 1
  • Wei-Min Liu
    • 1
  • Roman V. Agafonov
    • 2
  • Renee Otten
    • 2
  • Lien A. Phung
    • 2
  • Jesika T. Schilder
    • 1
  • Dorothee Kern
    • 2
  • Marcellus Ubbink
    • 1
    Email author
  1. 1.Leiden Institute of ChemistryLeiden UniversityLeidenThe Netherlands
  2. 2.Department of Biochemistry, Howard Hughes Medical InstituteBrandeis UniversityWalthamUSA

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