A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy
- 645 Downloads
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.
KeywordsRelaxation dispersion Lanthanide binding tags Protein dynamics Paramagnetic NMR Caged lanthanide NMR probe Adenylate kinase
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.
- Camacho-Zarco AR, Munari F, Wegstroth M, Liu WM, Ubbink M, Becker S, Zweckstetter M (2015) Multiple paramagnetic effects through a tagged reporter protein. Angew Chem Int Ed 54:336–339Google Scholar
- Hiruma Y, Hass MAS, Kikui Y, Liu WM, Olmez B, Skinner SP, Blok A, Kloosterman A, Koteishi H, Lohr F, Schwalbe H, Nojiri M, Ubbink M (2013) The structure of the cytochrome P450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography. J Mol Biol 425:4353–4365CrossRefGoogle Scholar
- Liu WM, Keizers PHJ, Hass MAS, Blok A, Timmer M, Sarris AJC, Overhand M, Ubbink M (2012) A pH-sensitive, colorful, lanthanide-chelating paramagnetic NMR probe. J Am Chem Soc 134:17306–17313Google Scholar
- Natrajan LS, Khoabane NM, Dadds BL, Muryn CA, Pritchard RG, Heath SL, Kenwright AM, Kuprov I, Faulkner S (2010) Probing the structure, conformation, and stereochemical exchange in a family of lanthanide complexes derived from tetrapyridyl-appended cyclen. Inorg Chem 49:7700–7709CrossRefGoogle Scholar
- Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371CrossRefADSGoogle Scholar