1. CHY 431 Biological Chemistry
Structure Determination by NMR
CHY 431 Biological Chemistry
Karl D. Bishop, Ph.D.
One Dimensional NMR
Two Dimensional NMR
Resonance Assignment Procedures
NMR demo programs … FREE!
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2. Acquiring the FID w z y x time voltage Receiver/transmitter
The receiver coil picks up the signal from the sample.
An analog-to-digital converter “reads” the voltage and
sends it to the computer for data storage.

3. One Dimensional NMR z Mo x w y x x x
90x
FID FT
time
frequency in ppm

4. Two Dimensional NMR
The two principle types of 2D NMR experiments are NOESY and COSY.
These can be either homonuclear, 1H-to-1H, or heteronuclear, 13C-to-1H.
A 2D data set can be thought of as a stack of 1D files.
Each 1D file is different from the next by a change in t1.
All other parameters are kept constant except the phase of the pulses.
Fourier transformation of each 1D in the t2 domain creates an interferogram.
The t1 domain is then Fourier transformed resulting in a 2D file with the
frequency in each dimension.
This 2D file will provide a map of all spin-to-spin correlations

5. COSY 2D Experiment w w The two dimensions are t1 and t2. t2 y x x x
preparation evolution acquisition
The two dimensions are t1 and t2.

6. COSY 2D Experiment · Typically there will be ~128-to-512FT
t1 = 750ms
t1 = 600ms
t1 = 450ms
t1 = 300ms t1
t1 = 150ms
t1 = 0ms t2 f2
Typically there will be ~128-to-512
t1 increments in a single 2D data file. t1

7. The Interferogram Interferogram FID t1 t1 f2 Contour plot.
2D plot of data
Bax and Morris, Jl. Magn. Res.,
42, (1981).

8. NOESY 2D Experiment Tm t2 90n 90n 90y t1
preparation evolution mixing acquisition
The two dimensions are t1 and t2.
t2 is the amount of time to acquire each FID.
t1 is an incremented time period or evolution time.
Tm is the “mixing time” during which the dipolar through-space coupling is allowed.

9. Polypeptide Spin System
7-10 ppm
ppm
Denotes spin systems in the individual residues
Denotes the aH-NH COSY connectivities
Denotes the sequential NOE connectivities
“NMR of Proteins and Nucleic Acids” Wuthrich, p131, (1986).

10. Sequential and Medium Range Distances
“NMR of Proteins and Nucleic Acids” Wuthrich, p118, (1986).

11. Nonsequential 1H-1H Distances in Proteins

12. Side Chain Coupling Patterns
diagonal peaks
COSY peaks
relayed COSY
+, *
“NMR of Proteins and Nucleic Acids”
Wuthrich, p136, (1986).

13. Side Chain Coupling Patterns
“NMR of Proteins and Nucleic Acids”
Wuthrich, p136, (1986).

14. Backbone Coupling in Peptides
“NMR of Proteins and Nucleic Acids”
Wuthrich, p119, (1986).

15. NMR Analysis of Ubiquitin
158 residues
1286 atoms
1305 bonds
Brookhaven 1A3S
4 alpha helical regions
1 or 2 b sheet residues.

16. Sample NMR Spectra of Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples courtesy of Ms. Tao Wang (Prof. David Yang's research group)

17. 2D COSY of Ubiquitin
Cavanaugh et al., 1996

18. NMR Analysis of Ubiquitin
Cavanaugh et al., 1996

19. Sample NMR Spectra of Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples courtesy of Ms. Tao Wang (Prof. David Yang's research group)

20. Sample NMR Spectra of Ubiquitin
obtained from Georgetown's 500 MHz Unity INOVA NMR Spectrometer
Samples courtesy of Ms. Tao Wang (Prof. David Yang's research group)