Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR

摘要

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear ¹⁵N-¹³C^α and homonuclear ¹³C-¹³C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped ¹³C^α resonances by strategically labeling the preceding residue. This method is highly complementary to ¹³C′i-1-¹⁵Ni-¹³C^αi and ¹³C^αi-1-¹⁵Ni-1-¹³C′i experiments to discriminate inter-residue spin systems at a minimal cost to signal-to-noise.