Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Poster Abstracts

32-POS Board 32 Reconstitution of Interactions between Bacterial Division Proteins in Lipid Coated Microbeads Marta Sobrinos Sanguino 1 , Silvia Zorrilla López 1 , Allen P. Minton 2 , Begoña Monterroso Marco 1 .Germán Rivas Caballero 1 . 1 Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 2 National Institutes of Health, Bethesda, MD, USA. A new quantitative method based on lipid coated microbeads using fluorescence spectroscopy has been optimized for the detection and characterization of interactions between proteins involved in bacterial division. In Escherichia coli , the proto-ring is the first multi-protein complex formed at the beginning of division. This complex consists of three essential proteins. Among them, we have focused on FtsZ, a GTPase that polymerizes in the presence of GTP, and ZipA, a membrane protein that anchors the cytoplasmic FtsZ to the membrane. Here we have analyzed the complexes of FtsZ and ZipA using lipid coated microbeads and other complementary methods to specifically address the impact of the density of ZipA receptors at the bilayer on the interactions with FtsZ. With this aim, silica microbeads have been functionalized by coating them with a mixture of phospholipids from E.coli containing different amounts of DGS-NTA lipids to which soluble mutants of ZipA, lacking the transmembrane domain, were anchored through a poly-histidine tag. The interaction between FtsZ and ZipA immobilized onto the microbeads at different densities was characterized through sedimentation, monitoring the fluorescence intensity of labeled FtsZ after microbeads pelleting. The effect of the nucleotide (GTP or GDP), on the interaction between immobilized ZipA and FtsZ was addressed. Preliminary results show that variations in the surface density of ZipA induce significant changes in the interaction with FtsZ. We also show that the fluorescence assay based on lipid coated microbeads can detect inhibitors of the FtsZ/ZipA interactions such as peptides derived from the C-terminal of FtsZ. Therefore, the lipid coated microbead assays proved to be useful for the characterization of interactions between division proteins and may also be applied for the systematic screening of inhibitors of these interactions, potentially useful for the development of new antibiotics.

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