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Protein in solution

Spectra. Radio waves. Principles of NMR Protein Spectroscopy. Frequencies and Spectra. 1 H 800 MHz 13 C 200 MHz 15 N 80 MHz. Protein in solution. B o 18.79 T. H z. H e. H e. H d. H d. H b. O. HN. H a. H b. NH. CH 3. H a. O.

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Protein in solution

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  1. Spectra Radio waves Principles of NMR Protein Spectroscopy Frequencies and Spectra 1H800 MHz 13C 200 MHz 15N 80 MHz Protein in solution Bo 18.79 T

  2. Hz He He Hd Hd Hb O HN Ha Hb NH CH3 Ha O 10 9 8 7 6 5 4 3 2 1 0 ppm Principles of NMR Protein Spectroscopy How would the 1H NMR spectrum appear? Ala Phe

  3. Hz He He Hd Hd Hb O HN Ha Hb NH CH3 Ha Actual O General n = gB0 (Hz) • is shielding constant can or n of nucleus ns = gB0 2p 2p -sgB0 (Hz) 2p Principles of NMR Protein Spectroscopy Chemical Shift - NMR is very sensitive to environment electronic, bonding Ala Phe small differences in n for each nucleus in molecule -same for 13C, 15N

  4. recall n = gB0 2p -sgB0 (Hz) - resonance condition (field + local) ns = gB0 2p 2p 8 d = e2 re dr 3me 0 Principles of NMR Protein Spectroscopy Chemical Shift B=Bo(1-) =d +p + m + r + e + s

  5. Hz He He Hd Hd Hb Ala Phe O HN Ha Hb NH CH3 Ha O 10 9 8 7 6 5 4 3 2 1 0 ppm Principles of NMR Protein Spectroscopy each nucleus has unique chemical shift similar for 13C, 15N 1H spectrum

  6. Principles of NMR Protein Spectroscopy Chemical Shift d = 106nsample - nref ppm 2877 nref nref = internal standard (0 ppm) 4.80 ppm DSS - dimethyl-silapentane-sulphonate 1H @ 14.09T nsample= 599.2933272 MHz DSS nDSS = 599.2904502 MHz d = 4.80 ppm Factors out B0 - d is same at any B0

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

  8. CH3 Aromatics Hb,g,d NH Ha 13C spectrum Cg,d CH3 Ca Cb 10 9 8 7 6 5 4 3 2 1 0 ppm 100 90 80 70 60 50 40 30 20 10 0 ppm Principles of NMR Protein Spectroscopy Chemical Shifts of Proteins In general nuclei found in “ranges” of chemical shift 1H spectrum

  9. Principles of NMR Protein Spectroscopy 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

  10. Hz He He Hd Hd Hb O HN Ha 7.03-7.11 Hb NH 8.19 CH3 Ha O 4.61 2.97, 3.00 4.26 1.37 8.39 Principles of NMR Protein Spectroscopy BioMagResBank actual chemical shifts for each amino acid are different dependence on placement in sequence and secondary structure Example Phe Ala

  11. n = gB0 gB0 2p 2p Principles of NMR Protein Spectroscopy Spin-spin Coupling     J A AB AB +J

  12. Principles of NMR Protein Spectroscopy Spin-spin Coupling 1H-1H 0-20 Hz 1H-13C 125-160 Hz 1H-15N ~ 93 Hz -CH3 -NH 2nI +1 lines 93 Hz 143 Hz 1H(15N) 13C(1H)

  13. Hz He He Hd Hd Hb O HN Ha Hb NH CH3 Ha O Principles of NMR Protein Spectroscopy Spin-spin Coupling 1H-1H 0-20 Hz 3J 3J = 6.98cos2 - 1.38cos +1.72  =  - 60˚

  14. Principles of NMR Protein Spectroscopy Other Coupling Dipolar Interaction D = || (3cos2 - 1) || = - 12h 42r3 2  Bo r12

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