3d and field gradients 12.ppt - university of...
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Extensions to 3Dand
Improving Efficiency with Pulsed Field Gradients
BCMB/CHEM 8190
2D NMR spectra can get very crowded
2D NOESY of Staph- Nuclease
156 AA
Solution: go to 3D
- - - C N C C N - - -
H
C
= =
- -
O O
H
H H
HH
- - - C N C C N - - -
H
C
= =
- -
O O
H
H H
HH
(HB)CBCA(CO)NH
HNCACB
3D experiments used for sequential resonance assignments: Can detect C in 3D by INEPT transfer from 13C to 15N then from 15N to 1H. Sequential connections from: HNCA plus HN(CO)CA
1D2D
3D
f3 (1H)
f1 (13C)
f2 (15N)
HN(CO)CA
Strategy for Constructing 3D Experiments:Combine various 2D Experiments
4D
combine more?
Cavanagh et al 1996
3D TOCSY-HSQC
Cavanagh et al 1996
TOCSY-HSQC for 15N-Labeled Ubiquitin
Cavanagh et al 1996
Pulsed Field Gradients: More Efficiency in Multidimensional Spectra
• Coherence selection using pulsed field gradients. J. R. Tolman & J. H. Prestegard, Concepts in Magnetic Resonance, 7, 247-262 (1995).
• Water suppression (WATERGATE), M. Piotto, V. Saudek & V. Sklenar, J. Biomol. NMR, 2, 661-665 (1992).
• Diffusion measurements. Altieri, Hinton & Byrd, J. Am. Chem. Soc., 117, 7566-7567 (1995).
Pulsed Field Gradients – How they Work
i
iB0(z)
B’
z
B0
time
G(z)
Magnetization vectors precess at different rates depending on G(z) and for each volume element. They dephase - net Mx, My = 0. Reversal of gradient refocuses magnetization.
Effects of Gradients can be Refocussed Application: Water Suppression
90x 90-y 180y 90-y
x y
z
a b x y
ab
zG1 G1
Resonance notaffected by 90refocuses
x y
z
b
a
x yb
a
zResonance affected by 90dephases (H2O)
x ya
b
z
x y
z
a
b
x y
z
b
a
1D 1H Water-Suppressed Spectrum Pf-Rubredoxin in 1H2O
ppm-2024681012
Translational Diffusion Constants for Macromolecules
• Determine aggregate size• Determine protein-protein interactions• Screen for bound ligands
• <(X1-X0)2> = nDt where D = kT/(6r)• Key: if molecule moves, field is different,
magnetization doesn’t refocus
r
90x 180y
gz gz
∆
•ln[S/S0] = -2g2D2(∆ - /3)
Stejskal and Tanner Pulse Sequencefor Diffusion Measurement
Diffusion Measurement Continued
• Measurements are limited by natural T2
• Improved sequence uses z storage(Altieri, Hinton and Byrd, 1995)
ln(S/S0)
g2
Large molecule
Small molecule
Coherence Selection Using Pulse Field Gradients
• H(r) = -kk [B0 + Bz(r)]Ikz
(in radians s-1 and neglecting chemical shifts)• Effects on product operators for a z gradient:
• Ikz -k Bz(z) Ikz Ikz
• Ikx = (Ik+ + Ik- )/2
• Ik+ -k Bz(z) Ikz exp[i k Bz(z) ] Ik+
• Ik- -k Bz(z) Ikz exp[-i k Bz(z) ] Ik-
• For linear gradients Bz(z) = Gz z• Observables are integrals over z – zero for Ik+ , Ik-
B’
z
B0
1H(I)
15N(S)90-x 180y 90y
90y
t1/2 t1/2 decouple180x 90-x 180x
180y 90-x 180x
t2
GG1
-G1G2
Gradient Selected HSQC
I+(t2) Integralz {S+(t1) exp[iN2G1z] exp[-iHG2z]}
Integralz {S+(t1) exp[i(N2G1z- HG2z)]}
I+(t2) finite only if N2G1 = HG2All 1Q proton transverse magnetization eliminatedNo phase cycling needed to suppress unwanted signals