Measurement of Angstrom to Nanometer Molecular Distances with ¹⁹F Nuclear Spins by EPR/ENDOR Spectroscopy

Abstract

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.

Application of W-band19F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

“…These approaches include electron–nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) techniques. 16 Recently, Marina Bennati and coworkers 17 proposed to use ENDOR in the W-band for a pair of labels (nitroxide and 19 F) for the distance measurements in biomolecules. 19 F atoms are exceptionally scarce in biological systems and have a high gyromagnetic ratio; thus, the ENDOR method allows for selective measurement of atomic distances up to 1.5 nm.…”

“…The approach proposed by Bennati et al 17,22 is based on the spin labeling concept from pulsed dipolar spectroscopy as applied to ENDOR via replacement of one of the nitroxides with a nuclear 19 F spin label to get access to molecular distances up to 1.5 nm. In their papers, they demonstrated the applicability of this approach on model systems and nucleic acid structures.…”

“…At high magnetic fields of 3.4 T (94 GHz, W band), the proton and fluorine Larmor frequencies are separated by 8.5 MHz, which prevents an overlap of 1 H and 19 F resonances. 17,22…”

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

“…CW dipolar broadening EPR method is used to obtain an intermediate distance range of 8-20 Å that can provide pertinent structural and dynamic information of the protein system [146]. Recently, a high-frequency (94 GHz) electron-nuclear double resonance (ENDOR) method using 19 F nuclei and nitroxide spin labels has been applied to obtain distance measurements up to~15 Å with an accuracy of 0.1-1 Å [147]. There are several important biological systems that have been studied using SDSL CW-EPR dipolar broadening techniques.…”

Trendbericht: Elektronen‐Paramagnetische‐Resonanzspektroskopie

“…Darunter die bei verschiedenen Feldwerten gemessen W-Band- 19 F-MIMS-ENDOR Spektren und deren jeweiligen Summen. aus 35) ungefähr 10 Å um das Elektronspin-Zentrum liefert dabei Strukturinformationen über die Bindungstasche. In diesem Zusammenhang sind die Arbeiten zu den biochemischen Metallcluster-Assemblierungswegen in den Hydro-und Nitrogenasen (Abbildung 5), 32) die mechanistischen Studien an den photosynthetischen Komplexen 33) und am Proton-gekoppelten Elektrontransfer in der Ribonukleotid Reduktase 34) zu nennen.…”