The relaxation induced dipolar modulation enhancement (RIDME) technique is applied at W-band microwave frequencies around 94 GHz to a pair of Gd(III) complexes that are connected by a rodlike spacer, and the extraction of the interspin distance distribution is discussed. A dipolar pattern derived from RIDME experimental data is a superposition of Pake-like dipolar patterns corresponding to the fundamental dipolar interaction and higher harmonics thereof. Intriguingly, the relative weights of the stretched patterns do not depend significantly on mixing time. As much larger modulation depths can be achieved than in double electron-electron resonance distance measurements at the same frequency, Gd(III)-Gd(III) RIDME may become attractive for structural characterization of biomacromolecules and biomolecular complexes.
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.
Lanthanide-induced enhancement of the longitudinal relaxation of nitroxide radicals in combination with orthogonal site-directed spin labeling is presented as a systematic distance measurement method intended for studies of biomacromolecules and biomacromolecular complexes. The approach is tested on a water soluble protein (T4-lysozyme) for two different commercially available lanthanide labels, and complemented by previously reported data on a membrane inserted polypeptide. Single temperature measurements are shown to be sufficient for reliable distance determination, with an upper measurable distance limit of about 5-6 nm. The extracted averaged distances represent the closest approach in LnIII-nitroxide distance distributions. Studies of conformational changes and of biomacromolecule association-dissociation are proposed as possible application area of the RE-based distance measurements.
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