1. Core C-2: NMR Center P.I. C. Allen Bush [bush@umbc.edu]bush@umbc.edu Director: Bruce Johnson [johnsonb@umbc.edu]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.

4. Free induction decay FID spectrum

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

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

16. Tocsy spectrum- pneumococcal CPS type 10F

18. Cosy spectrum- pneumococcal CPS type 10F

21. 1 H- 13 C HSQC spectrum of CPS of S. pneumoniae type 39 Anomeric signalsCentral region of spectrum

23. 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  g ) 2. insoluble solids 3. big protein (>50kD)

24. Practical Aspects Choice of Solvents – D 2 O for: Oligosaccharides, glycopeptides, polysaccharides D 2 O exchange: Lyophilize from D 2 O Other solvents (deuterated form): DMSO, CD 3 OD, CDCl 3 :

25. What can you see ? protons – 1 H bonded to carbon Amide protons in H 2 O (sometimes) Hydroxyl protons exchange rapidly in D 2 O

26. NMR Sample Requirements 1.Reasonably pure sample (~80%). Any impurity is readily obvious from the spectrum 2.High resolution NMR requires true solubility – cloudy solutions pose problems 3.Sample size: 1 micromole is good, 100 nM is OK. More sample provides more information, eg. 1 H- 13 C heteronuclear data in natural abundance 4.Isotope enrichment often used. eg. 13 C, 15 N in peptides or proteins

27. NMR Data Lab

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

34. [  PO  4  6GalNAc  1  3Rha  1  4Glc  1  6Galf  1  6GalNAc  1  3Gal  1  ] n Rha  1 2  [  PO  4  6GalNAc  1  3Rha  1  4Glc  1  6Galf  1  6Gal  1  3GalNAc  1  ] n Rha  1 2  1Gn (S. oralis 34) 2Gn (S. gordonii 38) 2G (S. mitis J22) RPS Type (strain) Antigenic RegionReceptor Region Structural Types of Receptor Polysaccharides (RPSs) 3G (S. oralis ATCC 10557) [  PO  4  6Gal  1  3Rha  1  4Glc  1  3Galf  1  6Gal  1  3GalNAc  1  ] n 26 (OAc) 0.33 [  1ribitol5  PO  4  6Galf  1  3Gal  1  6Galf  1  6GalNAc  1  3Gal  1  ] n 4Gn (S. oralis C104) [  3ribitol5  PO  4  6Galf  1  3Gal  1  6Galf  1  6GalNAc  1  3Gal  1  ] n 5Gn (S. oralis SK144)