title: Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins.
abstract: Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly (15)N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z's allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at "optimal" relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, United States.
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, United States. Electronic address: alex_nevzorov@ncsu.edu.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Nevzorov
mesh_terms: D000465:Algorithms; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals / Q000737:chemistry; D008051:Lipid Bilayers; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010742:Phospholipid Ethers / Q000737:chemistry
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D005609:Free Radicals; D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D010742:Phospholipid Ethers; D004134:Dimyristoylphosphatidylcholine
keywords: Free radicals; Longitudinal relaxation time; Magnetically aligned bicelles; Membrane proteins; OS-NMR; Oriented samples; PRE; Paramagnetic relaxation enhancement