Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 63, Issue 3, Pages 275-282Publisher
SPRINGER
DOI: 10.1007/s10858-015-9984-x
Keywords
Paramagnetic NMR; EPR; PELDOR; Lanthanide binding tags
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Funding
- Deutsche Forschungsgemeinschaft (DFG) [807, 902]
- DFG
- state of Hesse
- NSF [MCB 0744415]
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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-1 beta (IL1 beta) and measured H-1, N-15-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Delta chi-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 IL1 beta-S2R2 and the respective single-loop-LBT constructs IL1 beta-S2, IL1 beta-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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