Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.
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 (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-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 Gd(3+) 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.
Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438, Frankfurt Am Main, Germany.
Dominic
D
Barthelmes
Institute of Physical and Theoretical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438, Frankfurt Am Main, Germany.
Markus
M
Gränz
Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438, Frankfurt Am Main, Germany.|Department of Chemistry, Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Technical University Munich, Lichtenbergstrasse 4, 85747, Garching, Germany.
Katja
K
Barthelmes
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA, 02215, USA.
Karen N
KN
Allen
Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.
Barbara
B
Imperiali
Institute of Physical and Theoretical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438, Frankfurt Am Main, Germany. prisner@prisner.de.
Thomas
T
Prisner
Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438, Frankfurt Am Main, Germany. Schwalbe@nmr.uni-frankfurt.de.
Harald
H
Schwalbe
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011506:Proteins / Q000737:chemistry*
D028581:Lanthanoid Series Elements; D011506:Proteins
EPR; Lanthanide binding tags; PELDOR; Paramagnetic NMR