58 / 92
- 57 -
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.