Zusammenfassung
Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized because of a strongly reduced coherence time of the electron spins. Here membrane proteins, the maximum distance accessible is much ...
Zusammenfassung
Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized because of a strongly reduced coherence time of the electron spins. Here membrane proteins, the maximum distance accessible is much lower, we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each pi-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.
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