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Practical course „Molekulare und zelluläre Signaltransduktion“ NMR-spectroscopic investigation of proteins in solution Monika Beerbaum, Peter Schmieder AG NMR-Spektroskopie Leibniz-Institut für molekulare Pharmakologie Campus Berlin-Buch WS 2015/2106

NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

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Page 1: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course

„Molekulare und zelluläre Signaltransduktion“

NMR-spectroscopic investigation

of proteins in solution

Monika Beerbaum, Peter Schmieder

AG NMR-Spektroskopie

Leibniz-Institut für molekulare Pharmakologie

Campus Berlin-Buch

WS 2015/2106

Page 2: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

NMR-spectroscopic investigation of proteins in solution

For an explicit understanding of the function of biological active macromolecules

not only the chemical constitution but also the three dimensional structure, the

dynamical properties and the interaction with other proteins and small molecules

are of significant importance. The arrangement of functional groups in space and

their mobility have essential influence on the specificity of interactions for

example of ligands and proteins. Therefore besides functional studies

characterization of the structure, dynamic and interactions of biomolecules are

the prerequisite for an understanding of biological processes on an atomic level,

which is the basic for a rational manipulation of these processes in terms of drug

design.

NMR spectroscopy and X-ray crystallography are well established methods to

determine three dimensional structures on atomic scale. X-ray crystallography

determines structures in solid phase and requires adequate crystals. The

advantage is that there is no upper limit for the size of molecules that can be

handled.

With NMR structures can be determined in solution and in solid state. In solution

the size of the protein and the solubility are limiting, in solid state low sensitivity

and strong dipolar interactions. Nevertheless structure determination of proteins

up to 50 kDa is possible, even for membrane proteins.

Besides structure determination NMR is especial useful for investigating

dynamics of biomolecules and of even higher importance for the study of protein

ligand interactions. The latter are the actual key to the understanding of

biological processes and therefore of special interest for pharmacology.

Prerequisite for structural and dynamic investigations using NMR is not only the

synthesis of sufficient amounts of sufficient purity of the protein but also the

assignment of the resonances to the atoms of the protein.

This short practical course “NMR-spectroscopy” should give an insight into this

assignment procedure.

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Page 3: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 4: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 5: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 6: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 7: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 8: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 9: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

9/18

Page 10: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 11: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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Page 12: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

HNCA

1H

15N

1/2

90x 90y

90x180x

180x

1/2 2decoupling

13C

180x

180x

90x

90x

3/2 3/2

180x 90x

90x 180x

3/2-t2/2

t1/2 t1/2

2

90y

90x

1/2

180x

180x

1/2

180x

dec.3/2+t2/2

1 2 3 4H -> N -> Ca -> N -> H

H H

CC

C

O

O

N

HR

N

H

C

R H

N

1

23

4

Cai-1,i

HiNi

2

3

Ca-Experiment with 2 Ca-Signals per amino acid. Related Experiment with only one Ca-Signal (of amino acid i-1)

is HNCOCAi.

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Page 13: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

HNCOCAi

H H

CC

C

O

O

N

HR

N

H

C

R H

N

1

4

6

Cai-1

HiNi

1H

15N

90x 90y

90x180x

180x

decoupling

13C

180x

180x

90x

90x

180x

90x3/2-t2/2

t1/2 t1/2

90y

90x 180x

180x

180x

dec.3/2+t2/2

13C

180x

90x

90x

90x

90x 180x

180x

180x90x

180x

180x

180x 180x

1 2 3 4 5 6H -> N -> CO -> Ca -> CO -> N -> H

53 2

Ca-Experiment with one Ca-Signal per amino acid. Related Experiment with 2 Ca-Signals (of amino acid i and i-1) is

HNCA.

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Page 14: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

HNCO

1H

15N

1/2

90x 90y

90x180x

180x

1/2 2decoupling

13CO

180x

180x

90x

90x

3/2 3/2

180x 90x

90x 180x

3/2-t2/2

t1/2 t1/2

2

90y

90x

1/2

180x

180x

1/2

180x

dec.3/2+t2/2

1 2 3 4H -> N -> CO -> N -> H

H H

CC

C

O

O

N

HR

N

H

C

R H

N

1

23

4

COi-1

HiNi

CO-Experiment with one Ca-Signal per amino acid. Related Experiment with 2 CO-Signals (of amino acid i and i-1) is

HNCACO.

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Page 15: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

Vorlesung „Mehrdimensionale NMR-Spektroskopie-Grundlagen und

Anwendungen in der Strukturaufklärung"

HNCACO

4

COi,i-1

HiNi

1H

15N

90x 90y

90x180x

180x

decoupling

13CO

180x

180x

90x

90x

180x

90x3/2-t2/2

t1/2 t1/2

90y

90x 180x

180x

180x

dec.3/2+t2/2

13C

180x

90x

90x

90x

90x 180x

180x

180x90x

180x

180x

180x 180x

1 2 3 4 5 6H -> N -> Ca -> CO -> Ca -> N -> H

3

H H

CC

C

O

O

N

HR

N

H

C

R H

N

1

25

62

5 3

4

CO-Experiment with 2 CO-Signals per amino acid. Related Experiment with only one CO-Signal (of amino acid i-1)

is HNCO.

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Page 16: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

HNCACB

1H

15N

90x 90y

90x180x

180x

2decoupling

13C

180x

180x

90x

90x

3

180x 90x

90x 180x

3/2-t2/2

t1/2 t1/2

2

90y

90x

1

180x

180x

1

180x

dec.3/2+t2/2

1 2 3 4 5 6H -> N -> Ca -> CaCb -> Ca -> N -> H

Cabi-1,i

HiNi

Cab-Experiment with 2 Ca and 2 Cb-Signals per amino acid. Gives the same spectrum as a CBCANNH. Related Experiment with only

2 Cab-Signals (of amino acid i-1) are HNCOCACB or CBCACONNH.

180x

3

90x 90x 180x

3 3

H

HR

H H

CC

C

O

O

N

HC

R

N

H

CC H

N

1

2

2

3

3

4

45

56

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Page 17: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

HNCOCACB

1 2 3 4 5 6 7 8H -> N ->CO-> Ca ->CaCb-> Ca -> CO -> N -> H

Cabi-1

HiNi

Cab-Experiment with 1 Ca and 1 Cb-Signal per amino acid. Gives the same spectrum as a CBCACONNH. Related Experiment with 4 Cab-Signals (of amino acid i-1 and

i) are HNCACB or CBCANNH.

1H

15N

90x 90y

90x180x

180x

2decoupling

13C

180x 90x

90x

3

180x 90x

90x180x

3/2-t2/2

t1/2 t1/2

2

90y

90x

1

180x

180x

1

180x

dec.3/2+t2/2

180x

3

90x 90x 180x

3 3

13CO

180x 90x 90x 180x180x180x 90x

180x

H

HR

H H

CC

C

O

O

N

HC

R

N

H

CC H

N

1

2

4

3

5

6 7

8

180x

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Page 18: NMR-spectroscopic investigation of proteins in solutionschmieder.fmp-berlin.info/teaching/praktikum/pdf/beerbaum_schmieder… · NMR-spectroscopic investigation of proteins in solution

Practical course „Molekulare Pharmakologie und zelluläre Signaltransduktion : NMR-spectroscopy

Beerbaum/Schmieder AG Solution-NMR

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