Biophysical Society Thematic Meeting - October 25-30, 2015

Polymers and Self Assembly: From Biology to Nanomaterials Poster Session I

30-POS Board 30 Phalloidin Binds to MREB from Leptospira Interrogans

Szilvia Barko 1,2 , Emoke Bodis 1,2 , David Szatmari 1 , Robert C. Robinson 3,4 , Miklos Nyitrai 1,2,5 . 1 University of Pecs, Medical School, Pecs, Hungary, 2 János Szentágothai Research Center, Pecs, Hungary, 3 Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore, 4 Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 5 MTA-PTE Nuclear-Mitochondrial Interactions Research Group, Pecs, Hungary. MreB is a bacterial actin-like protein, which is a key player in the maintenance of cell shape and essential coordinator of the cell-wall synthesis. Although its sequence identity is low in comparison to eukaryotic actin, crystallography studies have shown that MreB and actin share a similar three-dimensional structure with conserved nucleotide-binding elements. The exact distribution of MreB in a bacterial cell remains uncertain due to potential mislocalization of the fluorescently tagged protein. Biochemical analyses have been confined mostly to T. maritima, B.subtilis, and E.coli MreBs, although the sequences and functions of MreBs can vary dramatically within a single species. One possible explanation for the limited number of described MreBs is the complicated purification of soluble functional protein. In our work we have purified and characterised MreB from Leptospira interrogans using denaturing purification protocol, which solved the previously mentioned problems in preparation. This MreB may carry novel structural and biochemical properties attributed to the special corkscrew shaped cell-type of Spirochetes. Here we show that MreB of L. interrogans is able to polymerise in vitro with association rates that depend on ionic strength and buffer conditions rather than the presence of nucleotides. Its cysteines can be labelled with Alexa 488 maleimide and the fluorescence intensity of the fluorophore changes during polymerisation. Surprisingly, it was found that L. interrogans MreB can be labelled also with fluorophore conjugated phalloidin. So far phalloidin binding has only been reported for actin. The observed MreB polymers are indistinguishable from that labelled with Alexa maleimide. Binding of phalloidin did not alter the biochemical properties of MreB. We envisage that the phalloidin staining of Leptospira interrogans MreB will provide a powerful experimental tool for the in vivo characterisation of the localisation and function of this important actin-like protein.

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