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Conformational Ensembles from Experimental Data
and Computer Simulations
Poster Abstracts
126
89-POS
Board 9
Conformational Flexibility of Multi-Domain Proteins Determined by Pulsed EPR
Denise Schuetz
1,3
, Sina Kazemi
2,3
, Kniss Andreas
2,3
, Oliver Mirus
4
, Eva M. Brouwer
4
, Peter
Günert
2,3
, Thomas Sommer
5,6
, Enrico Schleiff
4
, Volker Dötsch
2,3
, Thomas F. Prisner
1,3
.
2
Goethe-University, Institute of Biophysical Chemistry, Frankfurt, Hessen, Germany,
3
Goethe-
University, Center for Biomolecular Magnetic Resonance, Frankfurt, Hessen, Germany,
4
Goethe-University, Dept. of Molecular Cell Biology of Plants, Frankfurt, Hessen, Germany,
5
Max-Delbrück Center for Molecular Medicine, Berlin, Berlin, Germany,
6
Humboldt-Universität
Berlin, Institute for Biology, Berlin, Berlin, Germany.
1
Goethe-University, Institute of Physical
and Theoretical Chemistry, Frankfurt, Hessen, Germany,
Pulsed Electron-Electron Double Resonance (PELDOR / DEER)[1] spectroscopy in combination
with site-directed nitroxide labeling [2] is frequently used to gain distance restraints in the range
of 1.8 and 6 nm. [3] The distance and flexibility of the spin labeled protein domains are encoded
in the PELDOR time trace. Thereby, the intrinsic flexibility of the spin label itself could be an
obstacle for structural modelling, if the flexibility of the label is large compared to the flexibility
of the protein domains. Here, we present the investigation of two multi-domain proteins by the 4-
pulse DEER experiment [3]. First, the N-terminal polypeptide transport-associated (POTRA)
domains of anaOmp85 [4], is a rigid three domain protein giving well-defined PELDOR
restraints. These restraints are used for refining the x-ray structure [5], revealing a strong impact
of the spin label flexibility on the accuracy of structural refinement. Second, K48-linked
diubiquitin [6], is a highly flexible two-domain protein on which the spin label flexibility is of
minor impact. The recently developed 7-pulse Carr-Purcell PELDOR sequence [7] is applied to
extended polyubiquitin chains to study their high intrinsic flexibility. CP-PELDOR enables to
extend the PELDOR time window, thereby providing increased accuracy of the observable
distance distributions.
[1] A. Milov, et. al., Chem. Phys. Lett. (1984), 110, 67.
[2]W. Hubbell, et. al., Curr. Opin. Struct. Biol. (1998), 8, 649.
[3] M. Pannier
et.al., J Magn. Reson. (2000),142, 331.
[4] B. Clantin, et. al., Science 2007, 317, 957.
[5] P. Koenig, et. al., J. Biolog. Chem. 2010, 285, 18016.