期刊
JOURNAL OF PHYSICAL CHEMISTRY LETTERS
卷 6, 期 21, 页码 4331-4335出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jpclett.5b01933
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资金
- Collaborative Research Center Transport and Communication across Biological Membranes [807]
- priority program New Frontiers in EPR Sensitivity [SPP1601]
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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