Solids NMR of Biomolecules

BCMB/CHEM 8190

Techniques in Solids NMR

• Magic Angle Spinning (MAS)

• High Power Decoupling

• Cross Polarization (CP)

• Assignment Strategies

• Structural Information

Dipole-Dipole Interaction are Large:

E = (0/4)(( 1· 2)/r3 – 3(1· r)( 2·r)/r

5)

r = i rx + j ry + k rz = i r sincos + j r sinsin + k r cos

1

2

r

B0

x y

z

r

First Order QM Term (Iz1Iz2 ) is most Important

A doublet results – much like scalar coupling but large:

60,000 Hz for a 13C-1H pair, 250,000 Hz for 1H-1H .

Splittings are angle dependent – ranging from

-60,000 to +30,000 for 13C-1H. All possibilities

superimpose: The result is a powder pattern

Points at 90º on a sphere

are most abundant

D

Other Anisotropies in NMR

H = HCSA + HD + HQ...

All share the following property:

Solution: < 3 cos 2 '– 1 > = 0

Solids: (3 cos 2 ' – 1) ≠ 0

CSA powder pattern

Magic Angle Spinning

• All interactions can be written in terms of Y20() = (3cos2()–1)/2

• Y20() can be transformed to another frame using Wigner Rotation

elements: Y20() = 2

m=-2 D2

m0(’’,’’) Y2m (’,’)

• D2m0(’’,’’) = (4/5) Y2

m (’’,’’)

• With rapid averaging over ’’, all terms except Y20(’’) go to zero

• Selecting ’’ = 54.7, all interactions, regardless of ’ value, are zero

• (3cos2()–1) = (3cos2(’)–1) <3cos2(54.7)–1> = 0

Dipolar couplings

CSA = 0

Quadrupolar couplings

Z

X Y

’

’’

’’

High power decoupling

• MAS only works if rate >> D

– Maximum rate ~50 kHz, typical 10kHz

– High speed rotors ~ 1mm diameter

• Decoupling helps

– Easy in solution 13C-1H J = ~125 Hz

– Solids 13C-1H J + D = ~60 kHz

• Solution is to do both

Cellulose (10 minute spectra)

13C

What is this peak?

Cross Polarization Improves Sensitivity

selects for carbons with nearby protons

Magnetization transfer via dipolar coupling.

Hartman-Hahn:

gIBI = gSBS

Spinning Sidebands are Frequently Seen

When rotation rate is not >> anisotropies

Resonance position is modulated by rotation

Sidebands at the spinning frequency are produced

There are tricks that remove these:

TOSS – Total Suppression of Spinning Sidebands

180º pulses during rotor cycle dephases sideband

magnetization but preserves center band magnetization

Peptide

1,2-13C2-Gly

(9 minute spectra)

2D spectra use a variety of connectivities

here: proton driven spin diffusion

Aliphatic region of the 13C,13C CP MAS PDSD of Zn-MMP-12 (16.4 T,

11.5 kHz MAS frequency, 15 ms mixing time). (Balayssac, Oschkinat, 2007)

(C) Backbone walk for the A21–A28 stretch of residues in LC8 using three-dimensional NUS-PACC NCACB and NCOCA

experiments. The total experiment times are 27 and 9 h for the NCACB and NCOCA experiments, respectively. Reproduced

with permission from ref 6. Copyright 2012 American Chemical Society.

Published in: Si Yan; Christopher L. Suiter; Guangjin Hou; Huilan Zhang; Tatyana Polenova; Acc. Chem. Res. 2013, 46, 2047-2058.

DOI: 10.1021/ar300309s

Copyright © 2013 American Chemical Society

Experiments can parallel solution assignment strategies

Getting Distance Information

REDOR (rotational echo double resonance) Gullion T, Schaefer J (1989) J Magn Reson 81:196

1H

15N

90x 180y

r r r r r r

180y……. 180y……

• Refocuses the effects of MAS on dipolar coupling

• Two 180 degree pulses every rotor period

• Get phase modulation due at magnitude of dipolar coupling

Antibiotics & bacterial growth

Schaefer Laboratory, Washington University, St. Louis, MO

(A) 2D 13C–13C CORD sequences: (1) basic CORD; (2) CORDxix; (3) CORDxy4. These irradiation schemes are composites of

rotor-synchronized R2nv-symmetry sequences, R211 (ωrf = ωr), R212 (ωrf = ωr), R221 (ωrf = 1/2 ωr), and R222 (ωrf = 1/2 ωr).

Each R2nv element consists of two π pulses per n rotor periods. (B) 2D 13C–13C CORDxy4 spectra of U–13C,15N-LC8 (ωr =

40 kHz). Polarization transfer is uniformly efficient in both aliphatic and carbonyl regions of the spectrum.(11) Thioredoxin

Published in: Si Yan; Christopher L. Suiter; Guangjin Hou; Huilan Zhang; Tatyana Polenova; Acc. Chem. Res. 2013, 46, 2047-2058.

DOI: 10.1021/ar300309s

Copyright © 2013 American Chemical Society

Distances can be extracted using REDOR-like mixing

Angular dependence of dipolar interaction vectors

Separated local field spectra (PISEMA)

15N chemical shift

15N

– 1

H d

ipo

lar

co

up

ling

Gopinath, T.; Mote, KR.; Veglia, G (2013)

Sarcolipin in an oriented bicelle

Annual Reviews

2D solid-state NMR spectra of

uniformly 15N,13C-labeled Aβ1-40

amyloid fibrils. (a) 2D 13C-13C NMR

spectrum, obtained in a 14.1-T

magnetic field with 13.6-kHz

magic-angle spinning, using a

2.94-ms finite-pulse radio-

frequency-driven recoupling

(fpRFDR) sequence for spin

polarization transfer in the mixing

period. (b) 2D 15N-13C spectrum,

obtained with frequency-selective 15N-13C cross-polarization followed

by fpRFDR in the mixing period.

GPCR (CXCR1) structure in phospholipid bilayers Park, ….Opella, SJ., Nature 491:779 (2012)

SOLIDS NMR REFERENCES

Opella, S.J. (2013) Structure Determination of Membrane Proteins by NMR

Spectroscopy Annual Rev. Anal. Chem, 6: 305-328.

Yan, S, Suiter, CL, Hou, G, Zhang, H and Polenova, T. (2013) Probing Structure and

Dynamics of Protein Assemblies by Magic Angle Spinning NMR Spectroscopy,

Accounts Chem. Research 46: 2047-2058.

Gopinath, T.; Mote, KR.; Veglia, G (2013) Sensitivity and resolution enhancement of

oriented solid-state NMR: Application to membrane proteins, Prog. NMR

Spectroscopy 75: 50-68.

Solid-state NMR of matrix metalloproteinase 12: An approach complementary to

solution NMR (2007) Balayssac S, Bertini I, Falber K, Oschkinat, H, et al.

CHEMBIOCHEM 8 486-489.

Solid-State NMR Studies of Amyloid Fibril Structure (2011) Tycko R. Ann. Rev. Phys.

Chem. 62, 279-299.

Recent contributions from solid-state NMR to membrane protein structure and

function, Judge, PJ and Watts, A, (2011) Cur. Opin. Chem. Biol. 15, 690-695.