Browsing by Subject "Tubulin"

The bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.

My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.

I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.

The tubulin homolog FtsZ provides the cytoskeletal framework for bacterial cell division. FtsZ is an essential protein for bacterial cell division, and is the only protein necessary for Z-ring assembly and constriction force generation in liposomes in vitro. The work presented here utilizes structural and genomic analysis methods to investigate FtsZ function for cell division with three separate questions: (1) What is the function of the C-terminal linker peptide in FtsZ? (2) Are there interacting proteins other than those of the divisome that facilitate FtsZ function? (3) Do lateral contact sites exist between protofilaments in the Z ring, resulting in an organized Z-ring substructure?

The FtsZ protein has an ~50 aa linker between the protofilament-forming globular domain and the C-terminal (Ct) membrane-tethering peptide. This Ct linker is widely divergent across bacterial species, and has been thought to be an intrinsically disordered peptide (IDP). We have made chimeras where we have swapped the Escherichia coli IDP for Ct linkers from other bacteria, and even for an unrelated IDP from human &alpha-adducin. Most of these substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter -any IDP appears to work (with some exceptions). Length, however, was important: IDPs shorter than 39 or longer than 89 aa's had compromised function. We conclude that the Ct linker of FtsZ functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA and ZipA at the membrane. As a worm-like-chain, the Ct linker will function as a stiff entropic spring linking the constricting protofilaments to the membrane.

Previous work from our laboratory found that mutant and foreign FtsZ that do not normally function for cell division can function upon acquisition of a second site suppressor mutation, somewhere in the E. coli genome. We expect that some mutant or foreign FtsZ are partially functional for division in E. coli. As such, these FtsZ require another mutation that further enables their function. These suppressing mutations may reveal proteins interacting with FtsZ and the divisome, that have previously been unknown. In the present study, we have identified, via whole genome re-sequencing, single nucleotide polymorphisms that allow 11 different foreign and mutant FtsZ proteins to function for cell division. While we see a trend toward mutations in genes related to general metabolism functions in the cell, we have also identified mutations in two genes, ispA and nlpI, that may be interacting more directly with the cell division mechanism.

Finally, we have devised a screen to identify mutations in FtsZ that may be involved in lateral bonding between protofilaments. There are presently two proposed models of FtsZ substructure: the scattered or the ribbon model. A major difference between these models is that the scattered model proposed no interaction between adjacent protofilaments in the Z ring, while the ribbon model suggests that adjacent protofilaments are bonded laterally to create an organized substructure of aligned protofilaments. Our screen was designed to identify complementary surface-exposed residues that may be involved in lateral bonding. We initially identified two lateral contact candidate residues: R174, and E250 and mutated them to abrogate FtsZ function. We also mutated L272, which is known to make contacts across the protofilament interface, to look for compensating mutations in these contact residues. Using the screen, we identified a number of secondary mutations in FtsZ that can complement these initial loss-of-function mutations. While this screen has not yielded strong candidates for lateral bonding partners, it has emerged as a high-throughput method for screening large libraries of mutant FtsZ proteins in order to identify compensating mutation pairs.

FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?

One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.

The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.