EPR-based Gd(III)-nitroxide distance measurements were performed on a series of membrane-incorporated orthogonally labeled WALP23 polypeptides. The obtained distance distributions were stable upon the change of detection frequency from 10 GHz (X-band) to 35 GHz (Q-band). The α-helical pitch of WALP23 polypeptide could be experimentally observed, despite the flexibility of the two spin labels. The spectroscopic properties of Gd(III) ions and nitroxide radicals allow detecting both types of paramagnetic species selectively in different EPR experiments. In particular, this spectroscopic selectivity allows for supplementing Gd(III)-nitroxide distance measurements with independent checks of polypeptide aggregation and with measurements of the local environment of the nitroxide spin labels. All mentioned additional checks do not require preparation of further samples, as it is the case in the experiments with pairs of identical nitroxide spin labels.

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Longitudinal relaxation of nitroxide spin-labels has been measured for a membrane-incorporated α-helical polypeptide in the presence and absence of residual amounts of membrane-dissolved O2 and paramagnetic Dy(3+) ions. Such a model system, containing three different types of paramagnetic species, provides an important example of nonadditivity of two different relaxation channels for the nitroxide spins.

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.