Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery: Bridging Experiments and Computations - September 10-14, 2014, Istanbul, Turkey

Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session I

32-POS Board 32 Molecular Dynamics Comparison of E. coli WrbA Apoprotein and Holoprotein David Reha 1,2 , Dhiraj Sinha 1,2 , Balasubramanian Harish 3 , Zdenek Kukacka 4 , James McSally 3 , Olga Ettrichova 1 , Petr Novak 4 , Jannette Carey 3 , Rudiger Ettrich 1,2 . 1 Academy of Sciences of the Czech Republic, Nove Hrady, Czech Republic, 2 University of South Bohemia, Nove Hrady, Czech Republic, 3 Princeton University, Princeton, NJ, USA, 4 Academy of Sciences of the Czech Republic, Prague, Czech Republic. WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high- resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry [1]. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ~5 hours and resulting in complete conversion at the active-site methionine with partial conversion at second, heterogeneous sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidate the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly. [1] I Kishko, J Carey, David Reha, J Brynda, R Winkler, B Harish, R Guerra, O Ettrichova, Z Kukacka, O Sheryemyetyeva, P Novak, M Kuty, I Kuta Smatanova, R Ettrich, M Lapkouski (2013) 1.2 Å-resolution crystal structure of Escherichia coli WrbA holoprotein. Acta Crystallographica Section D: Biological Crystallography 69: 9. 1748-1757.

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