Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides

Abstract

Abstract. Membrane incorporated synthetic α-helical polypeptides labeled with Dy(III) chelate complexes and nitroxide radicals were studied by the inversion recovery (IR) technique and Dy(III)-nitroxide distances were obtained. A comparison of obtained distances with the previously reported Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) calibration data was performed and revealed reliability of the IR-based technique for the distance determination in membrane-incorporated biomacromolecules. The presented distance determination technique is 'spectroscopically orthogonal' to DEER-based distance measurements and can be potentially combined with DEER to study multiply spin-labeled biomacromolecules. The key steps of the data processing, the types of obtained distance information and the areas of possible application of the technique are discussed.

Combining NMR and EPR to Determine Structures of Large RNAs and Protein–RNA Complexes in Solution

“…Such EPR distance measurements rely on the dipole-dipole coupling that is revealed as a broadening in continuous-wave EPR spectra of nitroxide spin labels up to distances of about 20 Å (Rabenstein & Shin, 1995), by relaxation enhancement up to distances of about 30-40 Å ( Jäger, Koch, Maus, Spiess, & Jeschke, 2008;Lueders, Razzaghi, et al, 2013;Voss et al, 2001), and by pulse EPR techniques up to distances of about 80 Å ( Jeschke, 2012;Schiemann & Prisner, 2007). The data obtained by pulse techniques can be converted to distance distributions Jeschke, Koch, Jonas, & Godt, 2002), which is particularly useful in cases where the macromolecule or complex exists in several distinct conformations.…”

EPR Relaxation‐Enhancement‐Based Distance Measurements on Orthogonally Spin‐Labeled T4‐Lysozyme

“…[37, 38, 39] While several EPR studies were reported, where fast relaxing agents were naturally present in biomacromolecules,[40, 41, 42, 43, 44] the site-directed introduction of EPR relaxation agents and corresponding distance measurements have not yet been examined in detail. This approach is similar to the paramagnetic relaxation enhancement (PRE) approach in NMR, but due to the larger magnetic dipole moment of nitroxide radicals, as compared to any magnetic nuclei, the relaxation enhancement (RE) approach in EPR might access longer interspin distances than PRE.…”

“…For smaller monomeric molecules one or more paramagnetic centers can be synthetically built. [36, 39] Specific properties of a certain protein, as, for instance, protection of a labeling site by substrate binding, could be used for orthogonal labeling. [52] An approach that uses unnatural amino acids for nitroxide labeling[53] and a thiol-specific reaction for lanthanide labeling is currently the only tested systematic protocol of orthogonal chemo-selective spin labeling of large monomeric protein molecules.…”

“…[39] Here we aim to establish the RE approach as a systematic distance measurement tool in bio-EPR applications by addressing problems that arise in potential routine application of the approach. In particular, we discuss how to reduce the effort required to measure a single distance.…”

“…In particular, we discuss how to reduce the effort required to measure a single distance. Performance of the RE approach for membrane inserted[39] and soluble (this work) biomolecules is compared. The optimal experimental conditions, type of obtained distance constraints, and potential applications of the RE approach are discussed.…”