Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals

Abstract

Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.

Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins

“…Comparing with Cu 2+ -chelated complexes, paramagnetic ion chelated lipids eliminate the mobiliting of the ions avoiding RF heating that would damage the samples. More recently, Nevzorov’s group used PRE arising from free radicals to speed-up the acquisition of 2D OS-ssNMR of pf1 coat protein reconstituted in lipid bicelles [37, 39]. Here, we show that doping lipid bicelles with ~5% of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid copper salt (Cu 2+ -DMPE-DTPA) reduced 1 H T 1 relaxation rates of a membrane protein significantly, subsequently shortening the recycle delays and speeding up the acquisition of a 3D NMR spectrum used for sequential resonance assignment of a membrane protein amide backbone.…”

Membrane Topology of an Ion Channel Detected by Solid-State Nuclear Magnetic Resonance and Paramagnetic Effects

“…Because of reduced overall sensitivities by PRE effects from Mn 2+ or Dy 3+ , no 3D spectra with reasonable signal-to-noise ratios were acquired for KcsA samples. In the future, additional possibilities can be explored to enhance the overall signals of paramagnetic SSNMR membrane protein samples, such as fast MAS with low power decoupling and short recycle delays, − 1 H detection SSNMR with perdeuteration, ,, and dynamic nuclear polarization (DNP) enhanced SSNMR. , Other labeling schemes like glycerol labeling and selective reverse labeling could also be implemented to reduce the spectral complexity. Nonetheless, the technique of paramagnetic SSNMR of integral membrane proteins with chelator lipids can be useful to probe structural changes and topological shifts of residues related to the interactions between membrane proteins and ligands.…”