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  • Richard Waters posted an update 2 weeks ago

    We also present preliminary 1H detected 15N-1H heteronuclear correlation spectra of VDAC in lipid bilayers, illustrating the complementary and powerful approach of 1H detection in MAS NMR experiments. Comparing solution NMR and solid-state NMR assignments, some potential advantages within the present MAS study are noted, like the detection and assignment of a number of loop regions, that are typically hard to assign in detergent solubilized membrane proteins Title Loaded From File resulting from line broadening from conformational exchange.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptJ Biomol NMR. Author manuscript; available in PMC 2016 April 01.Eddy et al.Page2. Experimental SectionMaterials 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was obtained from Avanti Polar Lipids (Alabaster, AL). Octylpolyoxyethylene (Octyl-POE) was obtained from Bachem (King of Prussia, PA). All other non-isotopically labeled reagents had been obtained from Fisher. Isotopically labeled reagents applied for VDAC expression had been obtained from Cambridge Isotope Labs (Andover, MA). Preparation of VDAC/DMPC 2D crystals Protocols for expression, refolding, and purification of recombinantly expressed human VDAC have been based on the work of Malia and Wagner (Malia and Wagner 2007) and Hiller et al (Hiller et al. 2008b). 2D crystals had been ready based on the procedures published in Eddy et al.(Eddy et al. 2012), modified in the protocol initially described by Dolder et al.(Dolder et al. 1999). Isotopic labeling To facilitate the course of action of unambiguous assignments, various isotopically labeled samples had been ready with complementary labeling schemes. Along with a uniformly 13C, 15N labeled sample, many samples had been prepared with numerous all-natural abundance amino acids added before protein expression. For example, WHIFY-VDAC was prepared by adding 1 mM every all-natural abundance Trp, His, Ile, Phe, and Tyr along with 2 grams 13C glucose and 1 gram 15N ammonium sulfate. Similarly FLY-VDAC was ready by adding 1 mM each natural abundance Phe, Leu, and Tyr amino acids to a culture containing 13C glucose and 15N ammonium chloride. We refer to these sample s as “reverse” labeled. Moreover, numerous “forward” labeled samples had been ready by adding 1 mM 13C, 15N amino acids to media with all-natural abundance glucose and ammonium chloride. VPF-VDAC was prepared by addition of 1 mM 13C, 15N Val, Pro, and Phe, as well as 1 mM organic abundance every of all other 17 amino acids and two grams 12C glucose and 1 gram 14N ammonium chloride. [U-2H,15N,13C] VDAC was produced in media containing 99 D2O, 1 gram 15NH4Cl, and 3 grams 13C glucose. Protons had been introduced at exchangeable web-sites during protein purification. NMR spectroscopy For all 13C detected experiments, around 30 mg of 2-dimensional lipid crystals (20 mg VDAC and ten mg DMPC) had been packed into Bruker 3.2 mm MAS rotors and sealed with epoxy to stop sample dehydration. 1D spectra had been acquired by utilizing dipolar based cross polarization (CP) and INEPT (Morris and Freeman 1979) on an 800 MHz spectrometer at 20.0 kHz MAS and 283 K to investigate unique spectral functions of residues with distinctive dynamics. 2D homonuclear 13C-13C correlation spectra had been acquired with RFDR mixing at 12.five kHz MAS and 750 MHz 1H field strength on a custom constructed spectrometer (courtesy of Dr.

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