Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data and Computer Simulations

Poster Abstracts

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.

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