1. Protein in solution Radio waves 1 H 800 MHz 13 C 200 MHz 15 N 80 MHz Spectra Principles of NMR Protein Spectroscopy B o 18.79 T Frequencies and Spectra

2. Principles of NMR Protein Spectroscopy How would the 1 H NMR spectrum appear? HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe 10 9 8 7 6 5 4 3 2 1 0 ppm

3. Principles of NMR Protein Spectroscopy - NMR is very sensitive to environment HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe electronic, bonding small differences in for each nucleus in molecule   B 0 22 (Hz) s =  B 0 22 Actual General =  B 0 22 (Hz)  is shielding constant can or of nucleus -same for 13 C, 15 N Chemical Shift

4. Principles of NMR Protein Spectroscopy   B 0 22 (Hz) - resonance condition (field + local) s =  B 0 22 Chemical Shift B=B o (1-  ) =  B 0 22 recall  =  d +  p +  m +  r +  e +  s  d =   e 2 3m e r  e dr 0 8

5. Principles of NMR Protein Spectroscopy each nucleus has unique chemical shift HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH AlaPhe 10 9 8 7 6 5 4 3 2 1 0 ppm similar for 13 C, 15 N 1 H spectrum

6. Principles of NMR Protein Spectroscopy Chemical Shift 4.80 ppm  = 10 6 sample - ref ppm ref ref = internal standard (0 ppm) 1 H @ 14.09T sample = 599.2933272 MHz DSS  DSS = 599.2904502 MHz  = 4.80 ppm Factors out B 0 -  is same at any B  DSS - dimethyl-silapentane-sulphonate 2877

7. Principles of NMR Protein Spectroscopy Chemical Shift DSS 1 H @ 14.09T, DSS = 599.2904502 MHz 0 10 0 70 13 C @ 14.09T ref =  13 C * DSS  1 H =150.6913020

8. Chemical Shifts of Proteins 1 H spectrum In general nuclei found in “ranges” of chemical shift 10 9 8 7 6 5 4 3 2 1 0 ppm NH Aromatics HH H  CH 3 100 90 80 70 60 50 40 30 20 10 0 ppm CC CC CH 3 13 C spectrum C  Principles of NMR Protein Spectroscopy

9. Protein Database BioMagResBank http://www.bmrb.wisc.edu/ Reference Reference Information Chemical Shift Statistics Derived from the BMRB Database for: Restricted Set of Protein Chemical Shifts Full Set of Protein Chemical Shifts Restricted Set of DNA Chemical Shifts Full Set of DNA Chemical Shifts Restricted Set of RNA Chemical Shifts Full Set of RNA Chemical Shifts Principles of NMR Protein Spectroscopy

10. BioMagResBank actual chemical shifts for each amino acid are different dependence on placement in sequence and secondary structure Example HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH Ala Phe 8.19 7.03-7.11 4.61 4.26 1.37 8.39 2.97, 3.00 Principles of NMR Protein Spectroscopy

11. Spin-spin Coupling Principles of NMR Protein Spectroscopy     A AB =  B 0 22  B 0  22 +J J

12. Spin-spin Coupling Principles of NMR Protein Spectroscopy 1 H( 15 N) 1 H- 1 H 0-20 Hz 1 H- 13 C 125-160 Hz 1 H- 15 N ~ 93 Hz 2nI +1 lines 13 C( 1 H) 93 Hz 143 Hz -CH 3 -NH

13. Spin-spin Coupling Principles of NMR Protein Spectroscopy 1 H- 1 H 0-20 Hz HH HH HH HH HH NHNH O O HNHN HH HH CH 3 HH HH 3J3J 3 J = 6.98cos 2  - 1.38cos  +1.72  =  - 60˚

14. Other Coupling Principles of NMR Protein Spectroscopy D = || (3cos 2  - 1) Dipolar Interaction 2 || = -  1  2 h 42r342r3 BoBo r 12 