NMR-spectroscopic investigation of proteins in solution AG NMR-Spektroskopie Leibniz-Institut f£¼r molekulare

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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

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

    Beerbaum/Schmieder AG Solution-NMR

    3/18

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

    Beerbaum/Schmieder AG Solution-NMR

    4/18

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

    Beerbaum/Schmieder AG Solution-NMR

    5/18

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

    Beerbaum/Schmieder AG Solution-NMR

    6/18

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

    Beerbaum/Schmieder AG Solution-NMR

    7/18

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

    Beerbaum/Schmieder AG Solution-NMR

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

    Beerbaum/Schmieder AG Solution-NMR

    9/18

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

    Beerbaum/Schmieder AG Solution-NMR

    10/18

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

    Beerbaum/Schmieder AG Solution-NMR

    11/18

  • 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 2 decoupling

    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 4 H -> N -> Ca -> N -> H

    H H

    C C

    C

    O

    O

    N

    HR

    N

    H

    C

    R H

    N

    1

    2 3

    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.

    12/18

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

    Beerbaum/Schmieder AG Solution-NMR

    HNCOCAi

    H H

    C C

    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

    90x 3/2 -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 6 H -> N -> CO -> Ca -> CO -> N -> H

    5 3 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.

    13/18

  • 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 2 decoupling

    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 4 H -> N -> CO -> N -> H

    H H

    C C

    C

    O

    O

    N

    HR

    N

    H

    C

    R H

    N

    1

    2 3

    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.

    14/18

  • 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

    90x 3/2 -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 6 H -> N -> Ca -> CO -> Ca -> N -> H

    3

    H H

    C C

    C

    O

    O

    N

    HR

    N

    H

    C

    R H

    N

    1

    2 5

    6 2

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

    Beerbaum/Schmieder AG Solution-NMR

    HNCACB

    1H

    15N

    90x 90y

    90x180x

    180x

     2 decoupling

    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 6 H -> 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

    C C

    C

    O

    O

    N

    HC

    R

    N

    H

    C C H

    N

    1

    2

    2

    3

    3

    4

    4 5

    5 6

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  • Practical co