Core C-2: NMR Center P.I. C. Allen Bush [[email protected]][email protected] Director: Bruce Johnson...
-
Upload
ambrose-henderson -
Category
Documents
-
view
216 -
download
0
Transcript of Core C-2: NMR Center P.I. C. Allen Bush [[email protected]][email protected] Director: Bruce Johnson...
![Page 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/1.jpg)
Core C-2: NMR CenterP.I. C. Allen Bush [[email protected]]
Director: Bruce Johnson [[email protected]]
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
![Page 2: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/2.jpg)
NMR Spectrometer
An NMR instrument is composed of a magnet, a computer and a box of radios.
![Page 3: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/3.jpg)
![Page 4: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/4.jpg)
Free induction decay
FID spectrum
![Page 5: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/5.jpg)
![Page 6: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/6.jpg)
![Page 7: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/7.jpg)
![Page 8: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/8.jpg)
![Page 9: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/9.jpg)
Diaxial protons in -anomer > large J coupling valueEquatorial protons (gauche) in -anomer > smallJ coupling value
![Page 10: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/10.jpg)
![Page 11: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/11.jpg)
![Page 12: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/12.jpg)
![Page 13: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/13.jpg)
In the 2-d COSY spectrum the directly detected FID(blue) is recorded as the interval between the pulsesis incremented (the indirect dimension.)
![Page 14: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/14.jpg)
![Page 15: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/15.jpg)
![Page 16: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/16.jpg)
Tocsy spectrum- pneumococcal CPS type 10F
![Page 17: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/17.jpg)
![Page 18: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/18.jpg)
Cosy spectrum- pneumococcal CPS type 10F
![Page 19: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/19.jpg)
![Page 20: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/20.jpg)
![Page 21: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/21.jpg)
1H-13C HSQC spectrum of CPS of S. pneumoniae type 39
Anomeric signals Central region of spectrum
![Page 22: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/22.jpg)
![Page 23: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/23.jpg)
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)
![Page 24: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/24.jpg)
Practical Aspects
Choice of Solvents – D2O for:Oligosaccharides, glycopeptides, polysaccharides
D2O exchange: Lyophilize from D2O
Other solvents (deuterated form): DMSO, CD3OD, CDCl3:
![Page 25: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/25.jpg)
What can you see ?
protons – 1H bonded to carbon
Amide protons in H2O (sometimes)
Hydroxyl protons exchange rapidly in D2O
![Page 26: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/26.jpg)
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
![Page 27: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/27.jpg)
NMR Data Lab
![Page 28: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/28.jpg)
![Page 29: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/29.jpg)
![Page 30: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/30.jpg)
Martin-Pastor and Bush, Biochemistry, 39, 4674-4683 (2000)
![Page 31: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/31.jpg)
![Page 32: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/32.jpg)
![Page 33: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/33.jpg)
![Page 34: 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.](https://reader035.fdocuments.net/reader035/viewer/2022062519/5697bfe81a28abf838cb64b9/html5/thumbnails/34.jpg)
[ 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)