NMR-spectroscopic investigation. of proteins in solution
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1 Practical course Molekulare und zelluläre Signaltransduktion M-spectroscopic investigation of proteins in solution Monika Beerbaum, Peter Schmieder AG M-Spektroskopie Leibniz-Institut für molekulare Pharmakologie ampus Berlin-Buch WS 205/206
2 2/8 M-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. M 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 M 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. evertheless structure determination of proteins up to 50 kda is possible, even for membrane proteins. Besides structure determination M 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 M 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 M-spectroscopy should give an insight into this assignment procedure.
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12 A 2/ x 90 y 90 y 80 x /2 /2 2 decoupling 2 /2 / x 80 x 3 /2 3 /2 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 80 x dec. 80 x 80 x 3 t /2 t / > -> a -> -> i a i-,i i a-experiment with 2 a-signals per amino acid. elated Experiment with only one a-signal (of amino acid i-) is Ai.
13 Ai 3/ x 90 y 90 y 80 x decoupling 5 80 x 80 x 80 x 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 dec. 80 x 80 x 80 x 80 x 3 t /2 t /2 80 x 80 x 80 x 80 x 80 x > -> -> a -> -> -> a i- a-experiment with one a-signal per amino acid. elated Experiment with 2 i i a-signals (of amino acid i and i-) is A.
14 4/ x 90 y 90 y 80 x /2 /2 2 decoupling 2 /2 / x 80 x 3 /2 3 /2 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 80 x dec. 80 x 80 x 3 t /2 t / > -> -> -> i- -Experiment with one a-signal per amino acid. elated Experiment with 2 i i -Signals (of amino acid i and i-) is A.
15 A 5/ x 90 y 90 y 80 x decoupling x 80 x 80 x 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 Vorlesung Mehrdimensionale M- 80 x Spektroskopie-Grundlagen 80 x 80 x 80 x 90 und x 80 x Anwendungen in der Strukturaufklärung" dec. 80 x 80 x 80 x 80 x 3 t /2 t / > -> a -> -> a -> -> i i,i- i -Experiment with 2 -Signals per amino acid. elated Experiment with only one -Signal (of amino acid i-) is.
16 AB 6/ x 90 y 90 y 80 x 2 decoupling x 80 x 3 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 80 x dec. 80 x 80 x 80 x 80 x 3 3 t /2 t / > -> a -> ab -> a -> -> i ab i-,i i ab-experiment with 2 a and 2 b-signals per amino acid. Gives the same spectrum as a BA. elated Experiment with only 2 ab-signals (of amino acid i-) are AB or BA.
17 AB 7/ x 90 y 90 y 80 x 2 decoupling x 80 x 3 80 x 80 x 3 /2 3 /2 -t 2 /2 +t 2 /2 80 x dec. 80 x 80 x 80 x 80 x 3 3 t /2 t / x 80 x x x 80 x > ->-> a ->ab-> a -> -> -> i ab i- i ab-experiment with a and b-signal per amino acid. Gives the same spectrum as a BA. elated Experiment with 4 ab-signals (of amino acid i- and i) are AB or BA.
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