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Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

Abstract

We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured 1H, 15N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm3+-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein–protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1β-S2R2 and the respective single-loop-LBT constructs IL1β-S2, IL1β-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd3+ as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron–Electron Dipolar Resonance.

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Acknowledgments

We thank Dmitry Akhmetzyanov for helpful discussions. This work was supported by Deutsche Forschungsgemeinschaft (DFG) in collaborative research centers 807 and 902. H.S. and T.P. are members of the DFG-funded cluster of excellence: macromolecular complexes and BMRZ is supported by the state of Hesse. K.N.A and B.I. acknowledge the support of NSF Grant MCB 0744415.

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Correspondence to Thomas Prisner or Harald Schwalbe.

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D. Barthelmes, M. Gränz and K. Barthelmes have contributed equally.

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Barthelmes, D., Gränz, M., Barthelmes, K. et al. Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy. J Biomol NMR 63, 275–282 (2015). https://doi.org/10.1007/s10858-015-9984-x

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  • DOI: https://doi.org/10.1007/s10858-015-9984-x

Keywords

  • Paramagnetic NMR
  • EPR
  • PELDOR
  • Lanthanide binding tags