M z!=! N! 2! 2 I ( I +1)

Transcription

1 Advantages and disadvantages of a high magnetic field NMR and 3 C detection 202 Jan th Akihabara Satellite campus of Tokyo Metropolitan Univ. The 3 th RRR-workshop 20/2 Institute for Protein Research Osaka University Takahisa IKEGAMI 池上貴久

2 Advantages of a higher magnetic field NMR Higher sensitivity B 0 3/2 magnetic moment B 0 Larmor frequency B 0 noise B 0 /2 (S/N) 950MHz / (S/N) 600MHz = 2 M z!=! N! 2! 2 I ( I +)! 0!=!!"B 0 3kT B 0 Higher resolution in the direct-detection dimension (FID) The H- 5 N TROSY cross correlated relaxation effect A higher degree of alignment by anisotropic magnetic susceptibility

3 Nuclear magnetic moments nuclear spins static magnetic field, B 0 N energy Zeeman energy S β state ΔE = hv = ω α state

4 Comparison of particular regions in 2D NOESY spectra on Avance-I ms (t ) 239.6ms (t 2 ) Ns = 6 on Avance-III ms (t ) 240.3ms (t 2 ) Ns = 6

5 on Avance-I 800 on Avance-I 800 on Avance-III 950 on Avance-III 950

6 Detection of large proteins by TROSY J HN a 2D H- 5 N correlation spectrum J HN 5 N chemical shift (ppm) available to 800 kda? the maximum on GHz NMR? H chemical shift (ppm) H J HN N applicable to amide, methyl, and aromatic groups C α C o H N C α C o H N

7 B 0 cross-correlation between DD and CSA N S Sα Sx αβ ββ Iα dd instead of J Iβ N Iα βα S αα Ωs less shielded S! XX (! = 90 ) αβ ββ αα βα! XX TROSY-peak Ωs

8 when the molecule rotates by 90 (I-S on the horizontal) B 0 more shielded ββ! ZZ S αβ βα! ZZ (! = 0) αα Ωs magic angle ββ TROSY-peak N S Iα N S Sα Sx αβ βα Iβ Iα αα Ωs dd instead of J

9 The DD/CSA TROSY effect of 5 N- H depends on B TROSY!!!# $ R 2 CSA ( ) " R 2 ( DD) % & 2 chemical shift anisotropy 2 relaxation rate (/sec) dipole-dipole interaction 2 TROSY R 2 (CSA) 5 N R 2 (DD) 5 N- H N 系列 系列 2 系列 τ r = 20 ns (~50 kda), θ csa-dd = 5 dipole-dipole interaction x chemical shift anisotropy H resonance frequency (MHz)

10 The T relaxation of H N becomes slower in deuterated proteins. The dd relaxation rate with 2 Hα is ~/7000? A longer repetition-delay is required. DD Hn-,2 Hβ 5 DD Hn-,2 Hα DD Hn- 5 N DD Hn- Hn deuterated 系列 2 protonated 系列 CSA Hn H D α longitudinal auto-relaxation rate ρ (/sec) (assuming no cross-relaxation as in SOFAST HMQC) (500 MHz H) τ r =20 ns ( 50 kda) 5 N D β C α C β C o D β Good for TROSY, since the α and β states of H N are maintained for a long time.

11 Advantages and disadvantages of 3 C-direct detection (FID)! H!=!26.75!0 7!( T " s )! 3C!=!6.73!0 7!( T " s ) µ H :!2.79µ N µ 3C :!0.70µ N The gyromagnetic ratio of γ 3C is about /4 of γ H. µ N : the nuclear Borh magneton= J T Good The dipole-dipole T 2 relaxation is slow, particularly when 2 H is bound. Therefore, the 3 C detection provides narrower line-widths, being suitable for large and metallo-proteins. R( dd)!!!! 2 I "! 2 S " S( S +) Bad The sensitivity is low. Slower T relaxation requires a longer repetition-delay.

12 The T 2 relaxation by DD does not depend so much on B 0. R 2 DD (/sec) static magnetic field strength (MHz) R DD 2 =!2 2! I2! S! µ 0 $ # & 8r 6 " 4" % ( ) = 2 5 J!! c 2 { 4J ( 0 ) + J (! I '! S ) + 3J (! I ) + 6J (! S ) + 6J (! I +! S )} If 3 C FID is sampled for a sufficiently long time, the resolution represented by ppm is higher under a higher static magnetic field. τ c (ns) rotational correlation time +" 2! c 2 I ( 3 Cα) is observed. 3 Cα- Hα 2-spin system.09 Å DD alone is considered.

13 Disordered proteins exhibit a narrower distribution of H N chemical shifts. 寒川作 a cliff at 8.6ppm 2D H- 5 N HSQC unfolded folded 5 N chemical shift (ppm) H N chemical shift (ppm) H N chemical shift (ppm)

14 The hetero-nuclei like 5 N and 3 C have wider C.S. distributions even in unfolded proteins. unfolded 2D CONCO Pro ( 5 N ~34ppm) folded The projection along the H N dimension in 3D HNCO 5 N chemical shift (ppm) 3 Co chemical shift (ppm) 3 Co chemical shift (ppm)

15 Since H N is labile, its chemical exchange with water is a problem in H- 5 N HSQC of unfolded proteins at a high ph. at 293K The H N that is exchanging faster with water exhibits a smaller peak. at 30K

16 Low sensitivity in the 3 C detection 3 3 S N!!!Conc "! 2 exc "! obs " B " N scan start with H end (FID) with H B 0 : 600 MHz a normal probe end (FID) with 3 C start with 3 C B 0 : 950 MHz a cryogenic probe (inverse) = 00/ x higher concentration or 6 x data accumulation (NS) or starting with H (not applicable to 00% deuteration)

17 IPAP-process in 3 C detection D 5 N D 3 C α 3 C o D 5 N 3 C β in- phase an - phase

18 in- phase + an - phase in- phase - an - phase + -

19 virtual decoupling shifted by ± J CC 2

20 SQ CON with IPAP 25 ms y 3 Co 4J NCo t 2 4J NCo 4J NCo 4J NCo 5 N t AP IP IP AP 3 Cα H Gr 4J C!Co = 4.5ms N (ppm) H H 3 C 3 o C α C o 5 N C β mm [ 3 C, 5 N]- ubiqui n 80 3 Co (ppm) 70 provided from K. Furuita

21 The T 2 relaxation by CSA increases in proportion to B o2. R 2 CSA (/sec) static magnetic field (MHz) τ c (ns) rotational correlation time ( R CSA 2 =!!! II ") 2! 2 2 I B 0 J!! c 8 { 4J ( 0) + 3J (! I )} ( ) = 2 5 +" 2 2! c The CSA of 3 Co alone is considered.! xx =!5.6ppm,!! yy =!48.6ppm,!! zz = 40.6ppm

22 2D SQ CAN 22.2 ms y 3 Cα t 2 5 N t 2 H Gr M9- minimum medium for E.coli expression [2-3 C]- glycerol (or [,3-3 C]- glycerol) NaH 3 CO 3 D 2 O 5 N Proline can be detected, which has no amide H. IPAP is not needed in 3 Cα-FID. Sensitivity loss due to J CαCβ coupling does not occur. The T relaxation of 3 Cα can be enhanced by doping of paramagnetic metals. The sequential assignment of main-chains is possible with 2D COCA. D D 3 C α C β C o 5N C o Takeuchi, K. et al. (2008) J.Am.Chem.Soc. 30, 720. D D 3 C α C β

23 A tiny contribution of dd ( 3 Cα- Hα) to the 3 Cα T relaxation in a high field and high M.W. R DD (/sec) T =5 sec! static magnetic field (MHz) R DD =!2 2! I2! S! µ 0 $ # & 4r 6 " 4" % ( ) = 2 5 J!! c +" 2! c 2 2 { J (# I '# S ) + 3J (! I ) + 6J (! I +! S )} Is starting with H, having faster T, better than starting with 3 C? However, samples must be protonated. τ c (ns) rotational correlation time 3 Cα- Hα 2-spin system DD alone is considered. I ( 3 Cα) is detected.

24 The contribution from homonuclear dd ( 3 Cα- 3 Co) and dd ( 3 Cα- 3 Cβ) may become more dominant to the 3 Cα T relaxation for the increasing M.W. R (/sec) static magnetic field (MHz)! DD =!2 4! I! µ 0 $ # & 4r 6 " 4" % ( ) = 2 5 J!! c 2 +" 2! c 2 { J ( 0) + 3J (! I ) + 6J ( 2! I )} τ c (ns) rotational correlation time Deuterated 3 Cα is detected. Cross-relaxation is not considered as in 3 C SOFAST. The flip-back of 3 Co and 3 Cβ to z would be a 3 C version of SOFAST. The effect of deuteration is tiny (dd ( 3 Cα- Hα) accounts for ¼ of R ).

25 Unlike T 2, T relaxation by CSA does not so much depend on B o. R DD ( 3 Co- 3 Cα) R (/sec) R CSA ( 3 Co) static magnetic field (MHz) ( R CSA =!!! II ") 2! 2 2 I B 0 ( ) = 2 5 J!! c 3 +" 2! c 2 J (" I ) If 3 Cα is flipped-back to z, dd ( 3 Cα- 3 Co) becomes larger with the increasing M.W. τ c (ns) rotational correlation time 3 Co is detected.! xx =!5.6ppm,!! yy =!48.6ppm,!! zz = 40.6ppm

26 2D SOFAST CONCO? 25 ms y 3 Co 4J NCo t 2 4J NCo 4J NCo 4J NCo 5 N t 3 Cα AP IP IP AP H Gr Should we put to flip back 3 Cα to the z-direction?

27 B 0 Dipole-dipole coupling interaction when the relaxation of e- is slow(gd 3+, Mn 2+ ) Sx ββ e α e β αβ e α βα αα ωs ββ e β e α e α Sx αβ βα αα The large splitting is averaged out by the slow molecular rotation. ωs

28 Dipole-dipole coupling interaction B 0 Sx when the T relaxation of e- is fast(ni 2+ ) e α e β e α ββ Sx αβ βα ωs e β αα The large splitting is averaged out by the fast e- T relaxation rather than by the slower molecular rotation. ωs

29 What is more advantageous for 3 C-NMR over H-NMR u Since γ H is large, the dd relaxation is accordingly fast. By contrast, since γ 3C is small, the line width of 3 C is narrow enough to be detected even for high M.W. and metallo proteins. u Useful information can be obtained even from deuterated proteins having less number of H and from quaternary carbons. u For unfolded proteins 3 C exhibits a wider distribution of its chemical shift than H. u Water suppression is not required. No artifact comes from water. u There is no intensity loss due to exchange with water. Since H is labile, N H- 5 N- HSQC shows weak signals for H N that is exchanging with water fast. u 3 C tends to be more tolerant to chemical and conformational exchanges than H. u A higher sensitivity and resolution can be obtained in the FID detection at a higher magnetic field, since the T 2 relaxation by the dd interaction does not so much depend on the static magnetic field. u Signal loss due to high salt concentration is smaller?

30 Disadvantages in 3 C-NMR n Low sensitivity due to the small γ 3C. n J 3C-3C should be removed in the direct dimension. relaxation, a longer interscan- n Because of a long T delay is required. 寒川作 Future prospects of 3 C-NMR n Detection of 3 C is suitable for deuterated and large proteins under a high magnetic field. Since hydrogen is deuterated, the T 2 relaxation by dd is small, and thus the line width is narrower. H 5 N D α 3 C α 3 C o n Considering the current technology in terms of the sensitivity, however, the sequence starting with H N and ending with 3 C (FID) may be a compromise, which requires a shorter interscan-delay? D β 3 C β D β