protein nmr spectroscopy - biosupramol€¦ · protein nmr spectroscopy jana sticht ag freund, fu...

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Protein NMR spectroscopy

Jana Sticht AG Freund, FU Berlin

11.09.2014

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Applications of protein NMR

• dynamic information

NMR of proteins in solution

protein-ligand interactions

structural information

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What is special about proteins?

• they are big – often too big for standard NMR approaches

atoms in proteins

stable Isotope composition

(natural abundance)

Hydrogen (H)

1H (99.9885%) 2H (0.0115%)

Carbon (C)

12C (98.93%) 13C (1.07%)

Nitrogen (N)

14N (99.632%) 15N (0.368%)

12 12 10 8 6 4 2 0 -2

d1H [ppm]

methyl groups

aliphatic

Hα

aromatic

amide region

10kD protein

• problem: signal overlap overcome by isotope labeling of proteins expressed in bacteria

in combination with more dimensional spectra (nobel prize for Kurt Wüthrich 2002)

15NH4Cl

13C-glucose

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the HSQC (heteronuclear single quantum coherence) spectrum correlates covalently linked spins by magnetization transfer via J-coupling (JNH=92Hz)

magnetization transfer 15N to 1H

15N chemical

shift evolution

acquisition

N H

C

C

O

N

C

H

R2

H

R1

i i-1

O

C

H

1H-15N-HSQC spectrum – the “protein fingerprint”

magnetization transfer 1H to 15N

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1H-15N-HSQC spectrum – the “protein fingerprint”

• less peaks / better resolution • one peak per amide-group roughly one peak per residue (fingerprint!!)

N H

C

C

O

N C

H

R2

H

R1

i i-1

O

C

H

d(1H) [ppm]

d(1

5N

) [p

pm

]

1H-15N-HSQC of a 10kD protein

peak position (chemical shift) contains structural information

peak line-width delivers information on protein dynamics

which peak belongs to which residue? assignment needed!

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HNCO / HN(CA)CO - sequential assignment

N H

C

C

O

N C

H

R2

H

R1

HNCO signal for C‘ of NH(i-1)

i i-1

O

C

H

i

w H(i)

w NH(i)

w C‘(i-1)

w NH(i)

w H(i)

w C‘(i-1)

N H

C

C

O

N

C

H

R2

H

R1

HN(CA)CO signal for C‘ of NH(i) and NH(i-1)

i i-1

O

C

H

i

w H(i)

w NH(i)

w C‘(i-1)

w C‘(i)

w NH(i)

w H(i)

w C‘(i-1)

w C‘(1)

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HNCO / HN(CA)CO sequential assignment

w NH(i)

w H(i)

w C‘(i-1)

w C‘(i)

w NH(i-1)

w C‘(i-1) w C‘(i-2)

HNCO / HN(CA)CO

w H(i-1)

N H

C

C

O

N

C

H

R2

H

R1

HNCO signal for C‘ of NH(i-1)

i i-1

O

C

H

N H

C

C

O

N C

H

R2

H

R1

HN(CA)CO signal for C‘ of NH(i) and NH(i-1)

i i-1

O

C

H

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HNCA / HN(CO)CA sequential assignment

N H

C

C

O

N C

H

R2

H

R1

HNCA signal for Ca of NH(i) and NH(i-1)

i i-1

O

C

H

HN(CO)CA signal for Ca of NH(i-1)

N H

C

C

O

N C

H

R2

H

R1

i i-1

O

C

H w NH(i)

w H(i)

w Ca (i-1)

w Ca(i)

HNCA / HN(CO)CA

w H(i-1)

w Ca (i-1)

w Ca (i-2)

w NH(i-1)

very likely a glycine!

-x-Gly-

-x-Val/Ile/Thr-x-x-Gly-

-14Leu-15Thr-16Ala-17Ser-18Gly-

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assigned HSQC spectrum

available now: • assignment of NH signals in HSQC • backbone Ca and CO assignments

d(1H) [ppm]

d(1

5N

) [p

pm

]

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how to access this information




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d1H [ppm]

d15N

[p

pm

]

1H-15N-HSQC spectra:

one signal per NH-group

Gly16

Val12

Ile13

Gly4

Phe6

Mapping protein-ligand interactions

Val12

Ile13

d1H [ppm]

d15N

[p

pm

]

“fast exchange”

one sharp average resonance

d1H [ppm]

d15N

[p

pm

] “slow exchange”

two distinct resonances

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HSQC titrations allow to define binding sites and KD values of protein-ligand interactions

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0 1 2 3 4

ratio: [P]/[L]

com

bin

ed c

hem

ical

sh

ift

9GluH

20TyrH

29TrpH

53SerH

60TyrH

70TrpH

KD = 20 µM Dd(1

H,1

5N

) [p

pm

]

[P]/[L] d(1H) [ppm]

d(1

5N

) [p

pm

]

protein : ligand 1:0 1:0.7 1:1.3 1:6.7

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solution NMR



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collect structural information: secondary structure from backbone shifts

• Ca, Cb, CO assignments already contain secondary structure information

• Ca chemical shifts of a-helical or b-sheet regions differ from random coil values ∆𝛿𝐶𝛼 = 𝛿𝐶𝛼 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 − 𝛿𝐶𝛼 𝑟𝑎𝑛𝑑𝑜𝑚 𝑐𝑜𝑖𝑙

Dd(1

3C

a)[

pp

m]

protein sequence

protein + ligand A

protein + ligand B

secondary structure derived from crystal structure

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two published crystal structures – which is present in solution?

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68

56

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collect structural information: secondary structure from backbone shifts

Dd(1

3C

a)[

pp

m]

protein sequence

a-helix

56 68

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collect structural restraints: torsion angle

• quantitative J-correlation methods allow to determine 3J(HN-Ha) coupling constants

• cross-peak intensity ratio is correlated with coupling constant

Karplus correlation: 𝐽 𝜑 = 𝐴 𝑐𝑜𝑠2 𝜑 − 60 − 𝐵 cos 𝜑 − 60 +3 𝐶

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collect structural information: side-chain assignments

2D TOCSY (total correlation spectroscopy) correlation of entire spin system

3D TOCSY-HSQC

for bigger proteins

d(1H) [ppm]

d(1

H)

[pp

m]

HN

Ha

NH strip

Hb

Hg

t1 mixing pulse 1H

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collect structural restraints:

NOE secondary structure and distance information NOE (nuclear Overhauser effect) is due to through-space dipolar interaction between spins in

spatial vicinity (<5Å; NOE intensity ∝1

𝑟6)

2D NOESY

t1 tm 1H

for bigger proteins

3D 1H-15N-NOESY-HSQC intra- or intermolecular

cross-peaks

d(1H) [ppm]

d(1

H)

[pp

m]

NOEs to HN

NOEs to aliphatic hydrogens

NH strip

Ha

Hb

Hg

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NOE cross-peak intensity correlates with distance between atoms

strong 1.8-2.7 Å medium 1.8-3.3 Å weak 1.8-5.0 Å


NOE secondary structure and distance information

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F22

V34 M36

G20 F22

V34

d(1

H)[

pp

m]

d(1

H)[

pp

m]

d(1H) [ppm]

X

G20 M36


NOE secondary structure and distance information

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Structure determination

GYF-Smy2

(unpublished)

F

Y

+180 ° -180 °

+180 °

Ramachandran Plot

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solution NMR



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protein dynamics: peak line-width and relaxation

relaxation is dominated by rotational motion of the molecule:

small protein:

long FID sharp signal

big protein:

short FID broad signal

the peak line-width carries information on rotational motion of the molecule and internal dynamics

I(t) = I0 ei(wt) e-Rt

signal intensity (I) oscillating with Larmor frequency

exponential decay of the signal dominated by relaxation rate R2

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dynamics: quantitative information

qualitative information: broad signals indicate oligomerization, protein-protein interaction, conformational exchange

N H

quantitative information: regions of the protein displaying additional fast internal motion (ti, ps-ns) or conformational exchange (Rex, µs-ms) can be identified from relaxation rate measurements

ti

N

H

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relaxation time measurements

t

ma

gn

etization

0

equilibrium z magnetization Mz,eq

Mz(t) = Mz,eq - (Mz,eq - Mz(0)) e-t/T1

Inversion recovery to measure T1

1H t

Mxy(t) = Mxy(0) e-n(t-180-t) /T2

CPMG spin-echo to measure T2

1H t t

n

n(t-180-t)

ma

gn

etization

0

Mxy

longitudinal (T1) relaxation time transverse (T2) relaxation time

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dynamics: quantitative information

d(1H) [ppm]

d(1

5N

) [p

pm

]

1H-15N T1, T2, and hetNOE measurements allow to deduce protein backbone flexibility

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labeling strategies in big proteins

d(1H) [ppm]

d(1

5N

) [p

pm

]

uniform labeling

selective labeling

problems: • too many signals • broad lines due to fast relaxation

A125G B135G B54G A49G

B47Y

A104V

B90T

A45L

B41D

A55E

B157T

b-c

hain

a-c

hain

1H15N-TROSY-HSQC of deuterated 46kD protein

d(1H) [ppm]

d(1

5N

) [p

pm

]

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labeling strategies in big proteins d(1

5N

) [p

pm

]

uniform

amino-acid type specific 15N labeling 1H-15N-HSQC

d(1H) [ppm]

incorporation of

protonated amino acids

in deuterated background 1H-15N-NOESY-HSQC

Ala56Hb

d(1H) [ppm]

d(1

H)

[pp

m]

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labeling strategies in big proteins

d(1H) [ppm]

d(1

3C

) [p

pm

]

I,L,V-methyl group specific 1H-13C labeling 1H-13C-HSQC

105Leu

72Ile

6Val

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Acknowledgements

Marek Wieczorek

Zeina El Habre

Fabian Gerth

Stefan Klippel

Dr. Andreas Schlundt

Prof. Christian Freund

AG Proteinbiochemie, FU Berlin

Dr. Andreas Schäfer

Bettina Zeisig

NMR facility, FU Berlin

Dr. Peter Schmieder

Monika Beerbaum

NMR facility, FMP Berlin

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