1. Structure Determination by NMR CHY 431 Biological Chemistry Karl D. Bishop, Ph.D. One Dimensional NMR Two Dimensional NMR Resonance Assignment Procedures http://www.chem.vt.edu/chem-dept/hbell/simulation/VTNMR.html NMR demo programs … FREE! http://bmrl.med.uiuc.edu:8080/edusoft.html list os available NMR programs http://www.cm.utexas.edu/hoffman/index_gang.html

2. Acquiring the FID x  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. z y time voltage

3. One Dimensional NMR x MoMo 90 x y z xx x  FT frequency in ppm time FID

4. Two Dimensional NMR The two principle types of 2D NMR experiments are NOESY and COSY. These can be either homonuclear, 1 H-to- 1 H, or heteronuclear, 13 C-to- 1 H. 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 t 1. 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. 90 x 90 y preparation evolution acquisition t2t2 t1t1 COSY 2D Experiment The two dimensions are t1 and t2. x  y x x 

6. COSY 2D Experiment FT t 1 = 150  s t 1 = 300  s t 1 = 450  s t 1 = 600  s t 1 = 750  s t 1 = 0  s Typically there will be ~128-to-512 t 1 increments in a single 2D data file. t2t2 f2f2 t1t1       t1t1

7. The Interferogram f2f2 f1f1 f2f2 f1f1 t1t1 f2f2 t1t1 Interferogram FID 2D plot of data Contour plot. Bax and Morris, Jl. Magn. Res., 42, 501-05 (1981).

8. NOESY 2D Experiment The two dimensions are t1 and t2. t 2 is the amount of time to acquire each FID. t 1 is an incremented time period or evolution time. T m is the “mixing time” during which the dipolar through-space coupling is allowed. 90 n 90 y mm t2t2 t1t1 90 n preparation evolution mixing acquisition

9. Polypeptide Spin System “NMR of Proteins and Nucleic Acids” Wuthrich, p131, (1986). Denotes spin systems in the individual residues Denotes the  H-NH COSY connectivities Denotes the sequential  connectivities 7-10 ppm 3.5-6.0 ppm

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

11. Nonsequential 1 H- 1 H Distances in Proteins

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

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  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)