Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 March 2011
Nathaniel J., Traaseth , Gianluigi, Veglia
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? 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? resonances by strategically labeling the preceding residue. This method is highly complementary to 13C’i-1-15Ni-13C?i...
Graphical abstract
*Graphical abstract:**Highlights:*? Carbonyl-selective labeling in combination with frequency selective heteronuclear recoupling for assignments of crowded membrane protein samples ? The method discriminates overlapped [13]C[?] resonances by strategically labeling the preceding residue ? High complementarity to the current [13]C’i-1-[15]Ni-[13]C[?]i and [13]C[?]i-1-[15]Ni-1-[13]C’i experiments at a minimal cost to signal-to-noise.
Source:
Journal of Magnetic Resonance