Introduction into

solid-state NMR

Barth van Rossum, 21.02.2011

Barth-Jan van Rossum: Solid-State NMR

X-ray crystallography requires high-quality single crystal

à some systems notoriously difficult to crystallize…

typical solid-state NMR systems:

membrane-integrated receptors, aggregates, fibrils, intact organelles, (bio-)polymers...

Solid-state NMRno need for large well-ordered crystals or highly-purified proteins

works for immobilised proteins, no inherent limitation on complex size

à Solid-state MAS NMR very well-suited to study protein complexes that are difficult

to crystallize, insoluble or have tendency to aggregate.

Methods for structure determination (with atomic resolution)

rapid isotropic tumbling of the molecules

Solution-state NMRrequires rapid reorientation of soluble biomolecules

à Difficult to study proteins that either are large (> 50 kDa) or form

large complexes (aggregates, fibrils, membrane proteins)

Barth-Jan van Rossum: Solid-State NMR

proton chemical shift (ppm)

solid-state NMRno magic angle spinning no 1H-1H decoupling liquid-state NMR

solids-state NMR vs. liquid-state NMR

100 80 60 40 20 0 -20 -40 -60 -80

5,000 Hz

40,000 Hz

dipolar coupling between protons:

~ 120 kHz @ 1.0 Å~ 40 kHz @ 1.5 Å

Barth-Jan van Rossum: Solid-State NMR

solids-state NMR vs. liquid-state NMR

1 KW

720,844 mm3

(1 mW / mm3)

67 mm3

(15 W / mm3)

Solid-state NMR uses high-power RF pulses (1000 W) to manipulate the spins

5,000 Hz

15 mm3

(70 W / mm3)

Solid-state NMR is brute force…

Barth-Jan van Rossum: Solid-State NMR

tumbling rate

spectralcrowding

complex size versus protein size

solids and liquids:

à protein size determines spectral crowding

liquids:

à complex size determines tumbling rate

‘no inherent limitation on complex size’ : What does it mean?

Methods for structure determination (with atomic resolution)

SH3 domain62 residues (~7 kDa)

OmpG281 residues (~34 kDa)

solids:

à no upper limit for complex size

Barth-Jan van Rossum: Solid-State NMR

MAS mimics orientation averaging in liquids

by imposing a collective reorientation of all

molecules around a special axis

à “tumbling rate” not dependent on size

Magic-Angle Spinning (MAS)

what’s more:

- without MAS, only single crystals give a

high solid-state NMR resolution

- with MAS, you do not need crystals

However, it helps to have some sort of local

order …

tumbling rate

spectralcrowding

complex size versus protein size

drive

bearing

Magic-Angle Spinning (MAS)

Barth-Jan van Rossum: Solid-State NMR

What are ‘good’ MAS NMR samples?

For MAS NMR, you do not need crystals

However, it helps to have some sort of local order

non-ordered solid: each molecule (protein) in a different chemical environment

(identical spins experience different ‘local field’)

à broadening of the NMR lines

NAV(micro-crystalline)

QIY(lyophilized)

non-ordered

ordered

Barth-Jan van Rossum: Solid-State NMR

+

+

+

+

+

+

+

+

+

ppm

Barth-Jan van Rossum: Solid-State NMR

ppm

X-ray: long-range order required,

single-crystals required

MAS NMR: no long-range order required,

- short-range order sufficient

- non-crystallographic symmetries

Ordered system provides better NMR

resolution. But: no need for crystals in

the ‘classical’ sense

Barth-Jan van Rossum: Solid-State NMR

systems with high native

symmetry not good enough

for X-ray crystallography

- non-crystallographic

symmetry

- rotation symmetry

- combination of rotation and

translation: helical symmetry

X-ray crystallography

requires unit-cell that

repeats in all directions

and is related by

translation symmetry only

Barth-Jan van Rossum: Solid-State NMR

175180 ppm

70

65

60

55

50

45

40

35

30

25

20

15

10

5

70 65 60 55 50 45 40 35 30 25 20 15 10 ppm

70

65

60

55

50

45

40

35

30

25

20

15

10

5

LH2-complex: ~150 kDa9 protein units (in total 846 AA)+ pigments (BChl a and carotenoid)

Barth-Jan van Rossum: Solid-State NMR

“unshielded”

“shielded”

Electrons shield the nuclear spins from the external magnetic field

Chemical shift anisotropy (CSA)

Barth-Jan van Rossum: Solid-State NMR

solids: interactions depend on orientation of molecule

these interactions are called anisotropic

à limit resolution in NMR spectra of biological macromolecules

liquids: rapid random tumbling averages

anisotropic chemical shifts and couplings

Chemical shift anisotropy (CSA)

Barth-Jan van Rossum: Solid-State NMR

Chemical shift anisotropy (CSA)

axial symmetry

molecule perpendicular to B0 à maximum deshielding

molecule parallel to B0 à maximum shielding

non-axial symmetry

shielding is different in all three dimensions

spherical symmetry

shielding is similar in all three dimensions, CSA ~ 0

HCCH

H C C H

Barth-Jan van Rossum: Solid-State NMR

Chemical shift anisotropy (CSA)spherical symmetry

σ11 = σ22 = σ33

axial symmetry

σ11 = σ22 (or)

σ22 = σ33

non-axial symmetry

σ11 = σ22 = σ33

σ11 = σ22σ11,σ22 < σ33

σ22 = σ33σ22,σ33 > σ11

Barth-Jan van Rossum: Solid-State NMR

Chemical shift anisotropy (CSA)

Barth-Jan van Rossum: Solid-State NMR

drive

bearing

Magic-Angle Spinning (MAS)

Magic-Angle Spinning (MAS)

σiso

Anisotropic interactions can be suppressed using a

technique called magic-angle spinning

Barth-Jan van Rossum: Solid-State NMR

Magic-Angle Spinning (MAS)

“magic angle” : the angle between the body diagonal of a cube and the z-axis

à by rotation around this axis, a vector along z will cross the x and y-axes

54.7°

Barth-Jan van Rossum: Solid-State NMR

Magic-Angle Spinning (MAS)

note : for any rotation, one could construct a 1x1x1 cube around…

à however, the z-axis is ‘special’ (B0 direction!) and has to be one of the axes