Orientation Selective 2D-SIFTER Experiments at X-Band Frequencies

Frequency-correlated 2D SIFTER with broadband pulses at X-band frequencies can be used to determine the inter-spin distance and relative orientation of nitroxide moieties in macromolecules when the flexibility of the spin-labels is restricted. At X-band frequencies the EPR spectrum of nitroxides is governed by the strongly anisotropic nitrogen hyperfine coupling. For rigid spin-labels, where the orientation of the inter-connecting vector R correlates to the relative orientations of the nitroxide labels, the dipolar oscillation frequency varies over the EPR spectral line shape. Broadband shaped pulses allow excitation of the complete nitroxide EPR spectra. In this case, Fourier transform of the echo signal gives both fast and direct access to the orientation dependent dipole coupling. This allows determination of not only the inter-spin distance R, but also their mutual orientation. Here, we show the application of the frequency-correlated 2D SIFTER experiment with broadband pulses to a bis-nitroxide model compound and to a double stranded DNA sample. In both molecules, there is restricted internal mobility of the two spin-labels. The experimental results are compared to orientation selective pulsed electron double resonance (PELDOR) experiments and simulations based on a simple geometrical model or MD simulations describing the conformational flexibility of the molecules. Fourier transformation of the SIFTER echo signal yields orientation selective dipolar time traces over the complete EPR-spectral range. This leads to an improved frequency resolution and either to a reduced experimental measurement time or a larger span of frequency offsets measured compared to orientation selective PELDOR experiments. The experimental potential and limitations of the 2D SIFTER method for samples containing rigid spin-labels will be discussed.