title: 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.
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA.
authors.forename: Sophie N
authors.initials: SN
authors.lastname: Koroloff
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA.|Shaw University, 118 E. South Street, Raleigh, NC, 27601, USA.
authors.forename: Deanna M
authors.initials: DM
authors.lastname: Tesch
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA.
authors.forename: Emmanuel O
authors.initials: EO
authors.lastname: Awosanya
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA. alex_nevzorov@ncsu.edu.
authors.forename: Alexander A
authors.identifier: 0000-0001-9676-6139
authors.initials: AA
authors.lastname: Nevzorov
mesh_terms: D025561:Bacteriophage Pf1; D005609:Free Radicals / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D008565:Membrane Proteins / Q000737:chemistry*; D059629:Signal-To-Noise Ratio; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D005609:Free Radicals; D008565:Membrane Proteins; D014764:Viral Proteins
keywords: Magnetically aligned bicelles; Membrane proteins; Oriented-sample NMR (OS-NMR); Paramagnetic relaxation enhancement; Pf1; Repetitive cross-polarization; Sensitivity enhancement; Spectroscopic assignment