Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes

We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from <i>Escherichia coli</i> (<i>E. coli</i>), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photobleaching allows us to investigate Min proteins attaching and detaching from lipid coated gold nanorods with an unprecedented bandwidth of 100 ms time resolution and 1 h observation time. The long observation reveals small changes of the oscillation period over time. Averaging many cycles yields the precise wave profile that exhibits the four phases suggested in previous reports. Unexpected from previous fluorescence-based studies, we found an immobile static protein layer not dissociating during the oscillation cycle. Hence, NanoSPR is an attractive label-free real-time technique for the local investigation of molecular dynamics with high observation bandwidth. It gives access to systems, which cannot be fluorescently labeled, and resolves local dynamics that would average out over the sensor area used in conventional SPR

Polymorphism of FtsZ Filaments on Lipid Surfaces: Role of Monomer Orientation

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the <i>E. coli</i> FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation

The GTPase Activity of <i>Escherichia coli</i> FtsZ Determines the Magnitude of the FtsZ Polymer Bundling by ZapA <i>in Vitro</i>

FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in <i>Escherichia coli</i>. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ−ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl₂) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl₂). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published <i>in vitro</i> data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a <i>ΔzapA</i> strain <i>in vivo</i> and that it affected the interaction between FtsZ and ZapA <i>in vitro</i>