Core C-2: NMR CenterP.I. C. Allen Bush [bush@umbc.edu]

Director: Bruce Johnson [johnsonb@umbc.edu]

Newly Installed InstrumentationFour Bruker 3-channel Avance III consoles with

cryoprobe500, 600 MHz, 800 MHz and 950 MHzAll have identical Topspin 3.2 software

Data processing and analysis facilityData easily networked off-site

Funds available for training and exploratory work

NMR Spectrometer

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

Free induction decay

FID spectrum

Diaxial protons in -anomer > large J coupling valueEquatorial protons (gauche) in -anomer > smallJ coupling value

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

Tocsy spectrum- pneumococcal CPS type 10F

Cosy spectrum- pneumococcal CPS type 10F

1H-13C HSQC spectrum of CPS of S. pneumoniae type 39

Anomeric signals Central region of spectrum

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 solids3. big protein (>50kD)

Practical Aspects

Choice of Solvents – D2O for:Oligosaccharides, glycopeptides, polysaccharides

D2O exchange: Lyophilize from D2O

Other solvents (deuterated form): DMSO, CD3OD, CDCl3:

What can you see ?

protons – 1H bonded to carbon

Amide protons in H2O (sometimes)

Hydroxyl protons exchange rapidly in D2O

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. 1H-13C heteronuclear data in natural abundance

4. Isotope enrichment often used. eg. 13C, 15N in peptides or proteins

NMR Data Lab

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

[ PO46GalNAc13Rha14Glc16Galf16GalNAc13Gal1] n

[ PO46GalNAc13Rha14Glc16Galf16GalNAc13Gal1] n

Rha12

[ PO46GalNAc13Rha14Glc16Galf16Gal13GalNAc1] n

Rha12

1Gn (S. oralis 34)

2Gn (S. gordonii 38)

2G (S. mitis J22)

RPS Type (strain) Antigenic Region Receptor Region

Structural Types of Receptor Polysaccharides (RPSs)

3G (S. oralis ATCC 10557) [ PO46Gal13Rha14Glc13Galf16Gal13GalNAc1] n

2 6(OAc)0.33

(OAc)0.33

[ 1ribitol5 PO46Galf13Gal16Galf16GalNAc13Gal1] n 4Gn (S. oralis C104)

[ 3ribitol5 PO46Galf13Gal16Galf16GalNAc13Gal1] n 5Gn (S. oralis SK144)