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Advanced NMR Instrumentation and Capabilities for Structural Analysis

The NMR Center at UMBC boasts newly installed state-of-the-art Bruker 3-channel Avance III consoles operating at 500, 600, 800, and 950 MHz, all equipped with cryoprobes and utilizing Topspin 3.2 software. This facility offers data processing and analysis capabilities that are easily networked off-site. Funding is available for training and exploratory work. NMR can be used for analyzing structures, conformations, and ligand binding of various biomolecules, from oligosaccharides to small proteins. Our spectra include valuable insights into molecular characteristics.

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Advanced NMR Instrumentation and Capabilities for Structural Analysis

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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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