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Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR.

Jihyun KimRatnamala MandalChristian Hilty
Published in: Chembiochem : a European journal of chemical biology (2020)
Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σN,tail =-1.8±0.1 s-1  M-1 and σN,head =-0.5±0.3 s-1  M-1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.
Keyphrases
  • single molecule
  • amino acid
  • magnetic resonance
  • protein protein
  • optic nerve
  • high resolution
  • fatty acid
  • endoplasmic reticulum stress
  • binding protein
  • small molecule
  • computed tomography
  • endoplasmic reticulum