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Core C-2: NMR Center

Core C-2: NMR Center. P.I. C. Allen Bush [ bush@umbc.edu ] Director: Bruce Johnson [ johnsonb@umbc.edu ] Newly Installed Instrumentation Four Bruker 3-channel Avance III consoles with cryoprobe 500, 600 MHz, 800 MHz and 950 MHz All have identical Topspin 3.2 software

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Core C-2: NMR Center

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  1. Core C-2: NMR Center P.I. C. Allen Bush [bush@umbc.edu] Director: Bruce Johnson [johnsonb@umbc.edu] Newly Installed Instrumentation Four Bruker 3-channel Avance III consoles with cryoprobe 500, 600 MHz, 800 MHz and 950 MHz All have identical Topspin 3.2 software Data processing and analysis facility Data easily networked off-site Funds available for training and exploratory work

  2. NMR Spectrometer An NMR instrument is composed of a magnet, a computer and a box of radios.

  3. Free induction decay FID spectrum

  4. Diaxial protons in -anomer > large J coupling value Equatorial protons (gauche) in -anomer > small J coupling value

  5. In the 2-d COSY spectrum the directly detected FID (blue) is recorded as the interval between the pulses is incremented (the indirect dimension.)

  6. Tocsy spectrum- pneumococcal CPS type 10F

  7. Cosy spectrum- pneumococcal CPS type 10F

  8. Anomeric signals Central region of spectrum 1H-13C HSQC spectrum of CPS of S. pneumoniae type 39

  9. What can NMR do ? • Structure, conformation, ligand binding • Oligosaccharide, glycopeptides, polysaccharide, glycolipid • Small protein (<30kD), peptide • Ligand binding to (large) protein Cannot do 1. small sample (<100 mg) 2. insoluble solids 3. big protein (>50kD)

  10. Practical Aspects Choice of Solvents – D2O for: Oligosaccharides, glycopeptides, polysaccharides D2O exchange: Lyophilize from D2O Other solvents (deuterated form): DMSO, CD3OD, CDCl3:

  11. What can you see ? • protons – 1H bonded to carbon • Amide protons in H2O (sometimes) • Hydroxyl protons exchange rapidly in D2O

  12. NMR Sample Requirements Reasonably pure sample (~80%). Any impurity is readily obvious from the spectrum High resolution NMR requires true solubility – cloudy solutions pose problems Sample size: 1 micromole is good, 100 nM is OK. More sample provides more information, eg. 1H-13C heteronuclear data in natural abundance Isotope enrichment often used. eg. 13C, 15N in peptides or proteins

  13. NMR Data Lab

  14. Martin-Pastor and Bush, Biochemistry, 39, 4674-4683 (2000)

  15. [ PO46Gal13Rha14Glc13Galf16Gal13GalNAc1] n 2 6 (OAc)0.33 (OAc)0.33 Structural Types of Receptor Polysaccharides (RPSs) Antigenic Region Receptor Region RPS Type (strain) 1Gn (S. oralis 34) [ PO46GalNAc13Rha14Glc16Galf16GalNAc13Gal1] n 2Gn (S. gordonii 38) [ PO46GalNAc13Rha14Glc16Galf16GalNAc13Gal1] n 2  Rha1 2G (S. mitis J22) [ PO46GalNAc13Rha14Glc16Galf16Gal13GalNAc1] n 2  Rha1 3G (S. oralis ATCC 10557) 4Gn (S. oralis C104) [ 1ribitol5  PO46Galf13Gal16Galf16GalNAc13Gal1] n 5Gn (S. oralis SK144) [ 3ribitol5  PO46Galf13Gal16Galf16GalNAc13Gal1] n

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