3D NMR 3D NMR 3D NMR. Visualising 3D NMR spectra. strip plots. preparation mixing mixing t1 t2 t3 I S T I

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1 3D NMR 3D NMR Chris Waudby 3D NMR Visualising 3D NMR spectra preparation mixing mixing t1 t2 t3 I S T I 2 indirect dimensions, independently incremented evolution times Much longer acquisition than 2Ds (hours-days) SNR decreases by 2 for each extra dimension (quadrature detection, need to record separate sin and cos components) Longer sequences lower sensitivity strip plots

2 Effective long-range magnetisation transfer Optimisation of transfer efficiencies INEPT transfer vs relaxation 1.0 INEPT relaxation net transfer TINEPT 1 / 2J SNR ~ ns 1/2 conc B0 3/2 γex γobs 3/2 Michael Sattler transferred signal / sin( J )exp( R 2 ) INEPT relaxation Backbone 1 J and 2 J couplings The HNCO experiment J couplings proportional to gyromagnetic ratio (N.B. negative couplings involving 15 N) 2 J couplings very small! Michael Sattler CO chemical shifts very sequence dependent

3 HN(CA)CO HNCO / HN(CA)CO HN(CA)CO links HNi with C i-1 and C i HNCO links HN(CA)CO links 2 JNC < 1 Hz, so transfer via CA is required HNi with C i-1 HNi with C i-1 and C i HNCO / HN(CA)CO HNCA HNCA links HNi with CAi-1 (weak) and CAi (strong)

4 HNCA / HN(CO)CA CBCA(CO)NH HNCA links HNi with CAi-1 (weak) and CAi (strong) HN(CO)CA links HNi with CAi-1 HA/HB polarisation transferred to HN With deuterated protein, out-and-back HN(CO)CACB experiment must be used longer, less sensitive CBCA(CO)NH CBCANH HA/HB polarisation transferred to HN With deuterated protein, out-and-back HNCACB experiment must be used longer, less sensitive

5 CBCANH Relative sensitivity of 3D experiments CO/CA (i-1) CO/CA (i) CB (i-1) CB (i) HNCO HN(CA)CO HNCA HN(CO)CA CBCA(CO)NH CBCANH The assignment process Spin system typing CA and CB shifts are extremely useful for determining residue type Extent of CA and CB chemical shift dispersion is dependent on secondary structure formation bad for IDPs! Ser/Thr very easy to identify characteristic CB shifts (bonded to electronegative oxygen) Ala unique CB shift around 17 ppm Gly unique CA shift around 45 ppm and no CB resonances! Others are less clear, but usually can restrict to a few possibilities Prolines are only observed in i-1 experiments e.g. CBCA(CO)NH

6 alpha-synucein Sequence analysis identifying assignment checkpoints MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK ddfln5 Sequence analysis identifying assignment checkpoints KPAPS AEHSYAEGEG EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA LVKVFDNAPA EFTIFAVDTK GVARTDGGDP FEVAINGPDG LVVDAKVTDN NDGTYGVVYD APVEGNYNVN VTLRGNPIKN MPIDVKCIEG Sidechain assignment: HBHA(CO)NH Sidechain protons and magnetic equivalence Essentially identical pulse sequence to CBCA(CO)NH chemical shift encoded on 1 H rather than 13 C

7 Sidechain assignment: TOCSY 2D TOCSY experiment TOtal Correlation SpectroscopY Extension of HBHA(CO)NH wouldn t it be nice to get cross-peaks from ALL the spins in a sidechain? t1 τm DIPSI isotropic mixing t2 I 1,z! I 1,y! I 1,y cos(! 1 t 1 ) + other terms! I 1,z cos(! 1 t 1 ) eliminated by phase cycling at start of mixing period Isotropic mixing Isotropic mixing H = X i! i I iz + X i,j 2 J ij I i I j The spin-lock reduces the effective chemical shift difference between spins, increasing the efficiency of TOCSY transfer. = X i! i I iz + X i,j 2 J ij I iz I jz when ω >> J (weak coupling) The magnitude of this effect depends on the strength of the spin-lock relative to the frequency difference: DISPI spin-lock manipulates the effective Hamiltonian to remove chemical shift differences ( isotropic mixing ) so: H isotropic mixing = X i,j 2 J ij I i I j All spins are strongly coupled! Magnetisation will transfer between all spins in the spin system. Fundamentals of Protein NMR Spectroscopy, Rule & Hitchens

8 2D TOCSY experiment TOCSY transfer efficiency t1 τm DIPSI isotropic mixing t2 HN > X I 1,z! I 1,y! I 1,y cos(! 1 t 1 ) + other terms! I 1,z cos(! 1 t 1 ) eliminated by phase cycling at start of mixing period DIPSI cos(! 1 t 1 )I 1,z! X j sin 2 ( J e m ) cos(! 1 t 1 )I j,z isoleucine spin system Neglecting T2 relaxation! TOCSY transfer coefficient Cavanagh Optimising TOCSY mixing time CC(CO)NH Ubiquitin: 2D 1 H- 1 H TOCSY showing amide-aliphatic region lysine spin system 48 ms 83 ms 102 ms TOCSY transfer between aliphatic carbons Cavanagh

9 H(CCO)NH 15N TOCSY-HSQC TOCSY transfer between aliphatic carbons TOCSY transfer between protons HCCH TOCSY HCCH TOCSY Which chemical shifts to encode for 3D experiment? HcCH 1 H correlations to CH group hcch TOCSY transfer between carbons 13 C correlations to CH group

10 4D HCCH TOCSY 4D HCCH TOCSY δc δc δh δh Each point in 2D 1 H, 13 C spectrum is correlated with a second 2D 1 H, 13 C spectrum showing coupled spins Problem huge acquisition time! (can be addressed using non-uniform sampling ) Olejniczak et al (1992) J Biomol NMR 40 x 9 x 10 x 1024 points 6.4 days acquisition Olejniczak et al (1992) J Biomol NMR