These methods take advantage of large 1 J coupling constants

1. The number of protons yielding correlations in a 2D NOESY spectrum quickly overwhelms the space available on A 2D map. 15N labeling can help simplify the fingerprint region but not the aliphatic region

2. These methods take advantage of large 1J coupling constants

3. Backbone assignment via 1J couplings HNCA Start here – excite protons with a proton 90o pulse

4. HN(CO)CA

5. Slice from HNCA (at the 15N shift of I44, T14, R74..). Each pair of peaks correlates a Ca(i) and Ca(i-1) with the 1H and 15N shift of residue i. Slice from HN(CO)CA (at the 15N shift of I44, T14, R74..). Each pair of peaks correlates the Ca(i-1) with the 1H and 15N shift of residue i.

6. Stage 2. Sidechain assignments completed with HCCH-COSY and HCCH-TOCSY for example. The HCCH experiments provide connectivities of the aliphatic side chains of individual amino acid residues. Complete assignments can be obtained if the backbone assignments and the side-chain assignments can be connected via the 13Ca shifts.

7. An example. 13C shifts of Isoleucine We know the 13Ca shifts from the backbone assignment

8. Attempt to gain complete 1H, 15N and 13C chemical shift assignments. We can now resolve uncertainty in NOEs we observe. These 4 methyls would give an ambiguous network of possible NOEs. But suppose we knew that the 13C shift of the CH3 of Ile 1 was 9.3ppm and the CH3 of Ile 2 was 13 ppm.

9. Far larger proteins can now be tackled…44kDa Simian immuodeficiency virus (SIV) ectodomain used to fuse with host white blood cells

10. Types of Spin Relaxation • Longitudinal or spin-lattice relaxation (T1 )- recovery of longitudinal magnetization- establishment of thermal equilibrium populations- exchange of energy • Transverse or spin-spin relaxation(T2)-decay of transverse magnetization- no exchange of energy- increase of entropy

11. T1. Build up of longitudinal magnetization when field is switched on Mz (t) = Mzeq [1- exp{- (t-ton) / T1}] Spin-lattice relaxation time OR longitudinal relaxation time Equilibrium longitudinal magnetization

12. Inversion of longitudinal magnetization by π pulse 180o rotation about x-axis Recovery of longitudinal magnetization after π pulse 1 2

13. Rotational correlation time tc Simple theory of T1 small molecules tumble more quickly large molecules tumble more slowly rotational correlation time [in ns] approx. equal to 0.5  molecular mass [in kDa] 1 kDa = 1000 atomic mass units rotational correlation time Larmor frequency spin-lattice relaxation rate constant mean square amplitude of fluctuating fields

14. Precession of Transverse Magnetization Bo z z z xy plane y y y x x x Mx (t) = Mzeq sin(t) exp{-t / T2} Mx Time decay time constant =spin-spin relaxation time OR transverse relaxation time My (t) = -Mzeq cos(t) exp{-t / T2} oscillation at the Larmor frequency Time My The transverse magnetization components oscillate and decay

15. Transverse relaxation or T2 decay transverse magnetization is excited by first pulse along –y-axis transverse magnetization dephases due to field inhomogeneity during the interval t/2. “Black” vectors rotate faster than “grey” vectors

16. Problems with higher molecular weights and how to overcome them is the line-width in Hz at half peak height

17. Comparison of T1 and T2 rapid motion (small molecule non-viscous liquids), T1 and T2 are equal Slow motion (large molecules, viscous liquids): T2is shorter than T1.

19. Sensitivity of an NMR Experiment Signal to noise ratio Number of spins sample concentration Gyromagnetic ratio of excited spins isotope labeling Gyromagnetic ratio of detected spins out and back experiments Static magnetic field strength magnet “size” Number of scans measurement time Transverse relaxation time molecular weight Rattle page 46 and 47