Bio-Solid State NMR

EPJ Web of Conferences

30

, 03004 (2012)

DOI: 10.1051/epjconf/20123003004

© Owned by the authors, published by EDP Sciences, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which

Organized by

Thibault Charpentier

[email protected]

Patrick Berthault

[email protected]

Constantin Meis

[email protected]

džƉĞƌŝŵĞŶƚ ĂŶĚDŽĚĞůůŝŶŐ ŝŶ^ƚƌƵĐƚƵƌĂůEDZ

EŽǀĞŵďĞƌ ϮϴƚŚʹ ĞĐĞŵďĞƌ Ϯ

ŶĚ

_ϮϬϭϭ

/E^dEʹ ^ĂĐůĂLJ͕&ƌĂŶĐĞ

Hartmut Oschkinat

Leibniz Institut

für Molekulare Pharmakoligie

Germany

Biosolid state NMR

[03004]

Bio-SolidState

Hartmut Oschkinat

Leibniz-Institut für Molekulare Pharmakologie

Berlin

Saclay, 30th of November 2011

Solid-state NMR as a tool for ‘difficult’

areas of structural biology

Heterogeneous, filamentous systems:

Æ

cytoskelet-associated proteins

Æ

protofibrils

‘Dynamical complexes’

Æ

f-actin and associated proteins

Æ

focal adhesions

Æ

full-length aB-crystallin

NMR on membrane proteins

Bilayers and

liposomes:

Solid-state

NMR

Micelles:

Solution NMR

Spin Hamiltonian given as:

H

total

= H

Z

+

H

CS

+

H

J

+

H

D

+

H

Q

Zeeman interaction (

H

_Z

)

chemical shielding (

H

CS

)

,

J-coupling (

H

_J

)

dipolar coupling (

H

_D

)

quadrupolar coupling (

H

_Q

)

In solids: anisotropic contributions (<3cos

2

_{q-1> dependence); q is angle}

between the angle of the tensor element with respect to the applied field, B

_o.

For large (M

_r

» 30k) and slowly tumbling molecules (t

_r

> ~10's kHz), with all

orientations of the nucleus present at any one time, give rise to broad,

powder-like NMR spectra which are an envelope of the individual orientational

frequency dependent lines.

Experimental Observation

Orientation Studies: 15N labelling

B

₀

The Hamilton Operator of the Dipolar Interaction

and the Dipolar Alphabet

b

_IS

= −

μ

₀

γ

I

γ

S

!

4

π

r

_IS

3

The Hamilton Operator of the dipolar interaction

for 2 particles with spin 1/2 is given by

γ ≡

gyromagnetic ratio of the nuclei

!

≡

Planck

䇻

s constant

μ

≡

magnetic permeability

with

H

^

IS

DD

=

b

_IS

3

r

_IS

2

&

I

r

_IS

( )

( )

S

&

r

_IS

₋

I

&

S

&

­

®

¯

½

¾

¿

H

^

IS

DD

=

μ

0

γ

I

γ

S

!

4

π

r

3

3cos

2

_β

₋

₁

MAS

Speed,

ω

r

54.7

㼻

magnetic

field

54.7

㼻

Effects of magic angle spinning on solids

1

1

1

NMR Probes for solid state NMR

Practicalities of magic angle sample spinning

Sample contained in rotor 1.2 - 9mm diameter;

Usually fully hydrated; ~ 10 - 300nmoles of sample of interest; Usually

labeled with

13

_C,

15

_N,

2

_H,

19

_{F, etc}

Physics of Dipolar Recoupling

Typical magnitude in ppm of magnetic

interactions for various nuclei

Magic angle spinning solid state NMR

narrows anisotropically broadened spectra

Small (Mr ~ 3k) peptide in fluid lipid bilayers

Static NMR spectrum

Anisotropy broadened

(in chemical shift/

dipolar couplings)

13

_{C NMR}

_{magic angle 54.7}

_㼻

MAS NMR spectrum

Anisotropy averaged

by spinning

90

13

_C

Membrane protein structures by

solid-state NMR

Samples

and

Amyloid fibrils

3

roteins in a

micro-crystalline state

e.g. SH3 domain

2D crystals of

membrane proteins

e.g. OmpG

Ligand /cofactor bound

to a receptor

e.g. NT II + nAchR

Systems with short-range order can be

investigated by solid-state NMR

Reconstitution into lipid bilayers and

two dimensional crystallisation by dialysis

+

2D crystals

mixed lipid detergent micelles

refolded protein

detergent

LPR

1.0

3 k

- dialysis 6 days

1.0

45 k

- dialysis 8weeks

‘298 K’

400MHz

LPR

0.75

- dialysis 8weeks

45 k

0.5

900 MHz

α

-spectrin SH3 Domain

Barely soluble at pH 7

Protein structures by solid-state NMR

ASSIGNMENTS

Proton-driven spin diffusion (PDSD) experiment

to detect

13

_C-

13

_{C and}

15

_N-

15

_{N long-range}

correlations

1

_H

t

1

TPPM

2

t

CP

t

_mix

CP

t

_mix

: from 15 to 500

Identification of the single amino acids

0

20

40

60

80

threonine

α

β

γ

α

β

γ

0

20

40

60

80

0

20

40

60

80

NH

2

OH

O

HO

β α

γ

valine

0

20

40

60

80

α

β

α

β

γ γ

1, 2

γ γ

1, 2

0

20

40

60

80

0

20

40

60

80

NH

2

OH

O

β α

γ

1

γ2

70

60

50

40

30

20

70

60

50

40

30

20

V58C -C

γ

1

α

V58C -C

γ2

α

T24C -C

γ

β

T24C -C

γ

α

T24C -C

β

α

V58C -C

γ

1

β

V58C -C

γ2

β

_{V58C -C}

γ2

γ

1

V58C -C

β

α

13

C (ppm)

13

C(

pp

m

)

Chemical shift assignment

• Identification of

the single amino acids

3

3

‡

Sequential assignment

‡

Proton assignment

Chemical shift assignment

• Identification of

the single amino acids

3

3

‡

Sequential assignment

‡

Æ

Protonless MAS NMR requires thus a better

definition of the amino acid type prior to the

sequential assignment

13

_C,

15

_{N Backbone assignments:}

NCA NCACX (specific CP!)

NCO NCOCX

13

_C-PDSD

15

_N-PDSD

Resonance assignments using u-

13

_C

_,

15

_{N-labelled protein}

13

_{C Side chain assignments:}

13

_C-PDSD

13

_C-RFDR

1

_{H (}

13

_{C) assignments:}

1

_H-

13

_{C FSLG/PMLG}

3D

1

_H-

13

_C-

13

_{C FSLG-RFDR}

1

_{H (}

15

_{N) assignments:}

1

_H-

15

_{N FSLG/PMLG}

NCO and NCA spectra of (U-

13

_C,

15

_N)

_α

_{-spectrin SH3}

domains

[ppm]

110

115

120

125

130

G51

Y13

R21

V53

V58

V9

V23

T37

T24

T32

G28

A56

Y57

D40

N35

F52

Q50

D14

Y15

K39

V44

S36

K60

L12

L33

A55

L31

A11

L34, N38

K26

L10

L61

W42

W41

D29

M25

K43

E45

K59

E22

Q16

I30

K27

G51/Q50

Y13/L12

S19/K18

V58/Y57

V53/F52

T37/S36

N35/L34

V9/L8

A56/A55

Y57/A56

D40/K39

Q50/R49

G28/K27

T24/V23

D14/Y13

V23/E22

F52/G51

K59/V58, E45/V44

K43/W42

M25/T24, K39/N38

D29/G28

V44/K43

W42/W41

L10/V9

K26/M25

L34/L33

Q16/Y15

L31/I30

L33/T32

Y15/D14

T32/L31

I30/D29

E22/R21

K27/K26

W41/D40

S36/N35

L61/K60

N38/T37

K60/K59

A11/L10

L12/A11

A55/P54

R21/P20

R49/D48

E17/Q16

K18/E17

_E7,E17

15

N

13

C

13

C

S19

K18

170

160

180

190

65

60

55

50

45

[ppm]

D48/N47

D48

R49

L8

L8/E7

NCO

NCA

17.6 T, 278 K,

12 kHz MAS

PDSD, NCOCX and

NCACX spectra of

(U-

13

_C,

15

_N)

SH3 domains

[ppm]

[ppm]

15

20

25

30

35

40

45

50

55

60

65

70

75

15

20

25

30

35

40

45

50

55

60

65

70

75

[ppm]

110

115

120

125

130

135

170

180

190

T32/T32

T32

T32

T32

A55

A56

A11

A55

A56

A11

A56

A56

A56/A55

L33/T32

T32/L31

PDSD

NCOCX

PDSD

NCACX-PDSD

A55

A56

A11

T32

T32

T32

T32 CO

X

13

C

13

C

13

C

15

N

α

α

β

β

γ

γ

T32 F52 I30 T37 T24 K39/K60 K27/K60 I30 K60 K27 I30

T24 T32 T37 T32 T37 T24 S19 S36 K27 V53 I30 I30 I30 I30 I30 V9 V44 V23 V58 I30 K26 K59 K27/K43 K18/K60 K43 K43 K18 K18 R21/R49

K26 K26

K59 K59

K60 K60 K27 K27

V53 V53 V23 V23 V44 V44V9 V58 V58 V9 V53 V53 V9 V9 V44 V44 V58 V58 V23 V23 V9 V53 V58 V44 V23 K18 K18/L34 R21/R49 R21/R49 R21/R49 K43 K26/K59 L10

L8L31L61

L12 L33/L34

L10 L10 L10 L10 L10 L10

L8 L8 L8

L8 L8 L61 L61 L61 L61 L34 L33 L33/L34 L34 L33 L33 L12L12/L33 L12 L12 L12 L12 K18 P20 P54 P20 P20 P20 P54 P54 P20 D40 D48 N38 N35 D14 D29 Y13 F52 Y57 Q50/W42 Q50 Q16 Q16 E22 E22 E22 E17 E17 E7 E7/M25E45 E45 E45 E17 E7 M25 M25 W41 L61 L34 L34 K T32 T24 T37 T32 T24 T37 T32 T24 T37 E45 E45 E22 E7 E17 E22 Q16 Q50 Q50 Q50 E45 E22 E17 D40 D40 D14 D14 D29 D29 N35/N38 N35/N38 D48 S19 S36 T24_T37 T32 # § V53 V53 V58 V58 V9 V9 V23 V23 T37 V23 Q50 T24 K59 I30 I30 V44 V44 G51

F52 E22 K59 E45 I30 M25 K39 K27 K27 W41 R49 Y13 S19 V53 V58 V9 V23 T37 T24 G28 Y57 D40 N35 F52 Q50 D14 Y15 K39 V44 S36 K60 L12 L33 A55L31 A11 L61, N38 K26 L10 L34 W42 W41 D29, R49M25

K43 E45 _K59 E22 Q16 I30 K27 S36 T37 S19 P20 L61 N38 L34 K26

K60 K60 K60 Q16 L12 L31 A11 A55 L61 L10 R21 L34 L61/L34 D48 E17 K18 R21 E7, E17 L8 _E7 L33

H

N

C

1 15 13

ϕ

4

x

x

ϕ

ϕ

ϕ

1 2 3

y

-y

H

C

1 13

x

x

y

-y

NCOCX and NCACX spectra of (U-

13

_C,

15

_{N) SH3 domain}

17.6 T, 278 K

8 kHz MAS

[ppm]

110

115

120

125

130

135

15

20

25

30

35

40

45

50

55

60

65

70

75

[ppm]

110

115

120

125

130

135

15

N

13

13

_C

15

N

[ppm]

110

115

120

125

130

135

180

170

P20 Y15 Y15 K39 V44 V44 S36 S36 K60 K60 L12 L12

L33 A55 L33 L33

L31 L31 L31

L61, N38 L61, N38

L34

L34

L34

D14 D14

L10 W41L10 L10

D29, R49M25 D29 M25

K43 K43 E45 K59 E45/K59 E22 E22 Q16 Q16 I30 I30 W42 G51 Y13 Y13 S19 S19 V53 V53 V58 V58 V9 V9 V23 V23 T37 T24 T32 G28 A56 Y57 Y57 D40 D40 N35 N35 F52 F52 Q50 Q50 A11 K26 K26

K27 K18 W41_W42

K27 M25K39 R49

NCOCX

NCACX-PDSD

NCACX-BD

E7,E17 _E17 P54 P20 Q50 G51 V53 F52 G51/Q50 G51 F52/G51 F52 Q50 V53/F52 _V53 T37 N38 K39 β β β D48 D48

H

N

C

1

15

13

t t 1 2 TPPM TPPM x x CW x x y -y

TPPM CW TPPM

t 1

H

N

C

1

15

13

1

_H-

13

_{C-correlations by the frequency-switched}

Lee-Goldberg technique

1

_H

13

C

RAMPCP

t

2

+X

-X

+Y

+X

LG-CP

+Y

2

π

θ

m

−Δ

LG

+Δ

LG

2

π

/2

π

z

x

B

z

x

A

z

2D

1

_H-

13

_{C FSLG of}

13

_C-

15

_{N SH3 domain}

(11.5 kHz MAS)

Tcp=350

μ

s

Tcp=2.0 ms

400 MHz (9.4 T)

MAS : 8 kHz

T = 280 K

where discovered in 1896 in the eye lens

They are together a chaperone system for

β

- and

γ

-crystallin.

α

-crystallins (

α

A-crystallin and

α

B-crystallin)

Solid-state NMR of full-length

_α

B-crystallin

cataract

α

B-crystallin is distributed ubiquitously, acts as a

sHSP and mutants are involved in many diseases.

(e.g., multiple sclerosis, Alzheimers disease, cardiomyopathie)

aB crystallin forms dynamic, polydisperse oligomers

of 24 – 32 subunits, ~600kDa, with dimers as basic building blocks

conserved core domain

(

αααα

-crystallin domain, ~100 aa)

extension

tail

N-terminus

176 amino acids

alpha-B crystallin is a small heat shock protein

EM Structure at 3.6 nm

-

3D NCACX (N

_i

C

_i

), NCOCX (N

_i

C

_i-1

)

- 3D NCACB (N

_i

C

_i

), NCACBCX (N

_i

C

_i

)

- 2D

13

_C-

13

_{C correlations (PDSD)}

- J-decoupling

- Methyl-Filtering

3 Samples:

uniformly

13

_C,

15

_N

made from 1,3 -

13

_{C - glycerol}

made from 2 -

13

_{C - glycerol}

With help from Ponni Rajagopal,

Klevit Lab in Seattle!

Dataset/approach

171

171

53

57

173

57

173

57

L137N-C -C

α β

S136N-C -C

α β

S136N-S135C-Cβ

S136N-S135C-C

α

S135N-C -C

α β

L137N-S136C-L137Cα

S135N-C -T134C

α

α

S135N-C -T134C

α

S135N-T

134

C-C

α

S135N-T

134

C-Cβ

4

3

2

1,3G - NCOCX

U - NCACX

U - NCACB

1

U - NCOCX

U - NCACB

2G - NCACX

S76N-F75C-Cβ

E87N-C -Cα β

G102N-H101C-Cα

_S135N-T

₁₃₄

_C-C

_γ

₂

T134N-C -C

α γ2

T134N-C -C

α β

S139N-C -Cα β

61

T134N-C -C

α

L137N-S136C-C

β

U - NCOCX

U-NCOCX

U-NCACX

L137N-C -C

α γ

L137N-C -C

α δ

∗

a)

b)

c)

d)

e)

f)

g)

h)

i)

δ

15

_N(

_L137)

_{=122.9 ppm}

δ

15

L137)

ppm

N(

=122.9

δ

15

_N(

_L137)

_=122.9

_ppm

δ

15

S136)

ppm

N(

=118.3

δ

15

S136)

ppm

N(

=118.3

δ

15

_N(

_S135)

_=112.1

_ppm

δ

15

S135)

ppm

N(

=112.1

δ

15

S135)

ppm

N(

=112.1

δ

15

T134)

ppm

N(

=117.3

L137N-S136C-C

α

L137N-S136C-C

β

13

C

(pp

m

)

K103N-G102C-C

α

τ

mix

= 25 ms

τ

mix

= 75 ms

τ

mix

= 35 ms

τ

mix

= 2 ms

τ

mix

= 35 ms

τ

mix

= 2 ms

τ

mix

= 200 ms

τ

mix

= 25 ms

Structure of alpha-B crystallin in functional oligomers

Intermolecular N- and C-terminal

interactions are responsible for

oligomerisation

SAXS investigations

An oligomer model can be reconstructed, assuming tetrahedral

symmetry

T

I

P

T

I

conserved core domain

(

α

-crystallin domain, ~100 aa)

extension

tail

N-terminus

₍

_α

_{-crystallin domain, ~100 aa)}

conserved core domain

extension

tail

N-terminus

S59A

F118A

N78G-P86A

V LDV

N

KHF P

S

T S SS G

S L

T134K-T144R

D VL VN

T

Δ

P155-E165

PERTIPITREE

Monomer X

Monomer A

pH modulation of the chaperone binding site

pH 7.5

Solution NMR of flexible residues

The structure suggests that the activity of

_α

B-crystallin

is tightly connected to its oligomeric state. An intact

oligomer should not be active.

structural changes at pH 6.5, 9.0 and 4.5 suggest

regulation by pH

Conclusions

Protein structures determined by MAS solid-state NMR

1M8M, 62 aa

SH3 domain

Oschkinat

1RVS, 10 aa

Transthyretin

Jaroniec et al. 2004

1XSW, 38 aa

Kaliotoxin

Lange et al. 2005

2E8D, 4x 22 aa

beta2-microglobulin

Iwata et al. 2006

2JSV, 56 aa

GB1 (VEAN)

Franks et al. 2007

2JU6, 56 aa

GB1 (3D protons)

Zhou et al. 2006

2JZZ, 76 aa

Ubiquitin

Manolikas et al. 2008

2K0P, 56 aa

GB1 (CS)

Robustelli et al. 2008

2K9C, 152 aa

MMP-12 (PCS)

Balayssac et al. 2008

2KHT, 30 aa

alpha defensin HNP-1

Li et al. 2010

2KIB, 8x 7 aa

hIAPP

Nielsen et al. 2009

2KLR, 2x 82 aa

aB crystallin

Jehle et al. 2010

2KQ4, 56 aa

GB1 (3D TEDOR)

Nieuwkoop et al. 2009

2KRJ, 152 aa

MMP-12

Bertini et al. 2010

2NNT, 4x 31 aa

ww2 domain

Ferguson et al. 2006

2RLZ, 2x 85 aa

Crh dimer

Loquet et al. 2007

2RNM, 5x 71 aa

HET-s prion

Wasmer et al. 2008

2UVS, 38 aa

Kaliotoxin

Korukottu et al. 2008

2KWD

GB1 (TEDOR

Intermolecular)

Nieuwkoop et a. 2010

2KJ3, 3x 71 aa

HET-s prion

Quality of MAS solid-state NMR structures

Whatif quality Z-scores

Solid-state NMR

vs.

X-ray

vs.

solution NMR

SH

3

KT

X

KT

X

GB

1

GB

1

Crh

Ubq

GB

1

MMP1

2

HNP-1

GB

1

MMP1

2

GB

1

TTR

_WW

2

K3

β

2

HE

T

s

hiAPP

HE

T

s

aB

cr

ys

tallin

NMR on membrane proteins

ArtMP, an ABC transporter

ATP ADP+Pi ATP ADP+Pi

Arginine import system of

Geobacillus stearothermophilus

The holy grail:

dr

bearing

Magic-Angle Spinning (M

ATP ADP+Pi ATP ADP+Pi

ATP ADP+Pi ATP ADP+Pi

Reconstitution

2D Crystallisation

Preparation of ArtMP

Expression in

E.coli

Lipid extraction of

G. stearothermophilus

NMR-Spectra of ArtMP-2D-Crystals

$UW033<

ZLWKRXW $73

$UW037<3

ZLWK $73

_&

_&3'6'

_{0+].0LVFK]HLWPV}

&

SSP

_&SSP

&

SSP

1

_{H detection of perdeuterated samples, recrystallized from}

H

₂

O/D

₂

O = 1:9: SH3 with

1

_H,

2

_H,

13

_{C and}

15

_N

By Rasmus Linser, Vipin Agarwal, Veniamin Chevelkov,

Bernd Reif

R. Linser et al., Sensitivity Enhancement using paramagnetic

relaxation in MAS solid state NMR of perdeuterated proteins,

J. Magn. Res, subm.

Linewidths: 24

㼼

㼼

㼼

㼼

5 Hz

Use of standard pulse solution programs

ATP ADP+Pi ATP ADP+Pi

ATP ADP+Pi

ATP ADP+Pi

15

_N

15

_N

CP

INEPT

Reconstitution of ArtMP

ATP ADP+Pi ATP ADP+Pi

ATP ADP+Pi ATP ADP+Pi

15

_N

15

_N

15

_N

15

_N

CP

INEPT

Art(MP)

_15N

1

_{H detection of perdeuterated samples, recrystallized from}

H

₂

O/D

₂

O = 9:1

By Rasmus Linser, Vipin Agarwal, Veniamin Chevelkov,

Bernd Reif

Linewidths: 24

㼼

㼼

㼼

㼼

5 Hz

Optimum Levels of Exchangeable Protons in

Perdeuterated Proteins for Proton Detection

in MAS Solid-State NMR Spectroscopy:

A study at 24 kHz spinning

By the way: there are as many

exchangeable sites in the side chains of

the 20 aa as in the backbone!

Effect of

1

_{H% on}

1

_H-Linewidths

_{@ 24kHz-MAS}

1

_{H %}

_Average*

1

_{H FWHM (Hz)}

15

_{N FWHM (Hz)}

Average

10

19.0

±

3.3**

11.3

±

5.4

20

20.2

±

5.7

12.4

±

7.2

30

22.5

±

6.5

15.4

±

7.5

40

27.5

±

6.1

16.9

±

7.3

60

32.5

±

10.3

25.1

±

13.5

80

37.5

±

9.9

32.3

±

14.3

Another Issue !!

2D

1

_H-

15

_{N MAS NMR: Double-CP, 24 kHz, 275K, 400MHz}

Red: 20%

Black: 60%

Red: 20%

Black: 100%

Remaining & Dissappearing Signals

Remaining & Dissappearing Signals

S/N: CP

S/N per unit-time

1

_{H %}

1

_{H T1}

(s)

S/N Ratio (CP)

Average

S/N Ratio (CP)

per unit-time

10

4.28

18.9

±

4.7

21.1

20

2.58

33.5

±

11.4

48.4

30

2.13

41.1

±

16.4

65.3

40

1.67

44.6

±

22.6

80.1

60

0.85

26.4

±

15.7

66.5

80

0.76

18.3

±

13.9

48.7

100

0.68

11.1

±

6.3

30.9

Relaxation Delay: 7s.

Dynamic Nuclear Polarization

EWƉƌŝŶĐŝƉůĞ

ŶĞĞĚĞĚĂŵŽƵŶƚ

ϱŶŵŽů

ͬϮϱђů;

Ϭ͘ϮŵD

Ϳ

DNP Prinzip 1b

Gyrotron

controller

Gyrotron

produces

250 GHz

microwaves

Standard

400 MHz

NMR magnet

Cooling Cabinet

controls sample

temperature ~95K

3 pressurized

exchangers within

one dewar

LJŶĂŵŝĐEƵĐůĞĂƌWŽůĂƌŝƐĂƚŝŽŶʹ ƐŽůŝĚƐƚĂƚĞʹ EDZ

technique –

nACh-Receptor

dĞĐŚŶŝĐĂůŝƚŝĞƐ

Different sources of line broadening: radicals, freezing of

sample

Effect of the biradical concentration

Polarisation build-up in [s]

• Buildup behavior is

different for diff. Nuclei

•

1

_{H <}

13

_{C <<}

15

_N

• Deuteration increases the

IJ

_B

significantly

• Dipolar coupling & spin

diffusion is important.

^LJƐƚĞŵƐĂŶĚƚŚĞŵĞƐ

Systems and themes investigated in Berlin:

Membrane proteins without purification

Æ

Neurotoxin bound to nAchR in native

membranes

Æ

Mystic expressed in E.coli membranes

The nascent chain in ribosomes

Retinal in Drosophila eyes

The system

http://w

w

w

.dk

im

ages.c

om

/d

iscove

r/

p

revi

e

w

s/

824

/5

0200

72

.JP

G

http://w

w

w

.sevci

kpho

to

.com

/im

ages

/naj

a_

naj

a_2

.jpg

ďůƵĞ

͗ďŝŶĚŝŶŐƐŝƚĞƐĚƵĞƚŽ

<ƌĂďďĞŶĞƚĂů͕͘:D͕ϮϬϬϵ

ŚZ͗

ŵƵƐĐĂƌŝŶŝĐ͗ŵĞƚĂďŽƚƌŽƉŝĐ;'ͲWƌŽƚĞŝŶĐŽƵƉůĞĚ͖ďƌĂŝŶ͕ŚĞĂƌƚ͕ǀĞƐƐĞůƐͿ

ŶŝĐŽƚŝŶŝĐ͗ŝŽŶŽƚƌŽƉŝĐ;ůŝŐĂŶĚŐĂƚĞĚŝŽŶͲĐŚĂŶŶĞůƐͿ

ƉĂƌĂƐLJŵƉĂƚŚĞƚŝĐĂƵƚŽŶŽŵŝĐŶĞƌǀŽƵƐƐLJƐƚĞŵ͕ŶĞƵƌŽŵƵƐĐƵůĂƌũƵŶĐƚŝŽŶ

ƐŝŵŝůĂƌ͗'

Ͳ͕'ůƵƚĂŵĂƚͲZĞĐĞƉƚŽƌ͖ĨŝǀĞĚŝĨĨĞƌĞŶƚƐƵďƵŶŝƚƐ

EĞƵƌŽƚŽdžŝŶ//ďŽƵŶĚƚŽŶĐŚZ

room temperature

DNP

ZĞƐŽůǀĞĚƐƉĞĐƚƌĂ

ZĞƐŽůǀĞĚƐƉĞĐƚƌĂ

Ed//ĂƚŶĐŚZĐŽŵƉĂƌĞĚƚŽƌŽŽŵƚĞŵƉĞƌĂƚƵƌĞ

10 days

10 hours

dŚĞŶĂƐĐĞŶƚĐŚĂŝŶŝŶƚŚĞƚƵŶŶĞůŽĨƚŚĞƌŝďŽƐŽŵĞ

-the tunnel is ~100

‡

long

-with a diameter of ~10

‡

at a

constriction point

DsbA-SecM

vs.

Enolase-SecM

C7

CP 750 us

Mix 1ms

Exp.Time 3.8d

CP 750 us

Mix 1ms

Exp.Time 4d

DsbASecM:

MKKIW LALAG LVLAF SASAA FSTPV WISQA QGIRA GP

Enolase-SecM:

MSKIV KIIGR REIID SRGNP T FSTPV WISQA QGIRA GP

Ala

Ser

Thr

13C

13C

Ser - C

Į

:

G(B) = 5,5%

G(H) = 48,2%

G(C) = 46,3%

Ser - C

ββββ

:

G(B) = 6,8%

G(H) = 65,1%

G(C) = 28,1%

joint-probability:

Ser (C

Į

,C

ββββ

) :

P(B) = 0,83%

P(H) = 70,13%

Ser - C

Į

:

G(B) = 64,8%

G(H) = 0,1%

G(C) = 35,1%

Ser - C

ββββ

:

G(B) = 54,8%

G(H) = 0,14%

G(C) = 54,1%

joint-probability:

Ser (C

Į

,C

_ββββ

) :

P(B) = 69,2%

P(H) = 0,0%

P(C) = 30,83%

Ser - C

Į

:

G(B) = 14,9%

G(H) = 29,7%

G(C) = 55,4%

Ser - C

_ββββ

:

G(B) = 26,1%

G(H) = 21,4%

G(C) = 26,5%

Ser (C

Į

,C

ββββ

) :

P(B) = 9,8%

P(H) = 16,1%

P(C) = 74,1%

^ŽŵĞĞdžƉĞƌŝĞŶĐĞƐ͕ĐŽůůĞĐƚĞĚ

Samples

SH3

Ribosomes

Eyes

NT II

Kinesin

Mystic

Proline

sample amount

[nmol/rotor]

2000

16

2

40

50

30

99

sample

appearance

solid

wet pellet

wet pellet

solid

wet pellet

wet pellet

solid

added glycerol

[

μ

l]

15

5

5

6

15

5

15

S/N (carbonyls)

194

33

23

86

73

62

67

enhancement

39

16

17

10

11

30

38.5

T1 H [s]

3,00

2,30

1,40

1,90

0,60

1,20

1,90

ĐŬŶŽǁůĞĚŐĞŵĞŶƚƐ

ϭϱ͘Ϭϱ͘ϮϬϭϮ

ƌŶĞ>ŝŶĚĞŶͶ ϴϰ

DNP (NMR)

Arne Linden

Sascha Lange

Ümit Akbey

Trent Franks

Barth- Jan van Rossum

Wet Lab

Nils Cremer

Anne Diehl

and all others

Colleagues at MIT

Bob Griffin

Torsten Maly

Alexander Barnes

Björn Erzelius

• Oschkinat et. al.:

– Dr. Bart van Rossum

– Dr. Trent Franks

– Dr. Ümit Akbey

– Sasha Lange

– Arne Linden

– Dr. Anne Diehl

• Reif et. al.:

– Prof. Bernd Reif

– Rasmus Linser

– Vipin Agarwal

– Venjamin

• .

DNP-Magnet measurements

Thanks to ...

DNPers

(

D

ashing

N

itrogen

P

ush

ers

)

NMR/structure calculation

B. van Rossum

Trent W. Franks

S. Jehle

S. Lange

A. Linden

V. Lange

R. Kü

hne

J. Pa

uli

M. Fossi

N. Schröder

P. Schmieder

Preparation SH3 and aB-c:

K. Rehbein

OmpG:

M. Hiller

W. Kühlbrandt (Fra)

Ö. Yildiz (Fra)

K. Vinothkumar

NeurotoxinII/nAchR:

L. Krabben

C. W

eis

e, FUB

F.

Huch

o, FUB

WW-

F

ibrils

:

A. Fersht, Cam.

Protein structure determination by magic-angle spinning

solid-state NMR

Nascent chain:

Bernd Bukau, Heidelberg

Anna Rutkowska, Heidelberg

Melanie Rosay, Billerica

Leo Tometich, Billerica

Bob Griffin, MIT

Thorsten Maly, MIT

aB-crystallin: