# A glowing sensor to watch a bacterial division protein at work > FtsZ is the bacterial cousin of tubulin and forms the ring that divides a bacterium; it splits GTP into GDP and phosphate as it works. Source: https://khushisingh.in/research/phosphate-biosensor-ftsz/ Author: Khushi Singh (khushisingh.in). Licence: CC BY 4.0 for text and original illustrations, unless a credit says otherwise. Evidence labels used on this site: established, strong evidence, emerging, hypothesis, debated. Where a page has Simple, Curious and Expert versions, all three follow. --- [Research](https://khushisingh.in/research/) · Completed · 2025–2026 IISER Pune · Structural Biology of Cytoskeletal Systems lab, Prof. Gayathri Pananghat Proteins from: [*Escherichia coli*](https://khushisingh.in/research/model-system/#escherichia-coli) · [*Spiroplasma melliferum*](https://khushisingh.in/research/model-system/#spiroplasma-melliferum) The question How can I watch, in real time, how fast a bacterial cell-division protein uses its fuel? FtsZ is the bacterial cousin of tubulin and forms the ring that divides a bacterium; it splits GTP into GDP and phosphate as it works. A phosphate-binding protein labelled with a fluorescent dye (PBP-MDCC) brightens when it binds phosphate, so it reports enzyme activity continuously. I prepared and labelled the sensor and used it to study the GTPase kinetics of FtsZ from Spiroplasma melliferum, a protein family targeted for new antibiotics. [The basics: making proteins](https://khushisingh.in/explore/05-making-proteins/) ## Why ask this? Almost every bacterium divides using **FtsZ**, a protein that assembles into a ring (the *Z-ring*) at the middle of the cell. FtsZ is a distant cousin of tubulin, the protein that builds our own cells’ skeleton, and it only burns its fuel, GTP, once it has assembled into filaments. Because the Z-ring is essential for bacteria and differs from anything in our cytoplasm, it is a long-standing **target for new antibiotics**. (evidence: established) Measuring how fast FtsZ uses GTP tells me how its filaments behave. The classic trick is to detect the **phosphate** released, one molecule per GTP used. *[Here the web page has an interactive 3D model; open the source URL to use it.]* ## The tool: a protein that glows for phosphate The *E. coli* phosphate-binding protein snaps shut around phosphate like a Venus flytrap. Swap one amino acid for a cysteine, attach the dye **MDCC**, and the dye brightens when the trap closes. This turns an invisible molecule into **light you can measure every few seconds** (Brune et al. 1994). (evidence: established) *[Here the web page has an interactive 3D model; open the source URL to use it.]* 1. **1 · A protein that changes shape** E. coli phosphate-binding protein: two lobes that close on phosphate 2. **2 · Add one cysteine** Alanine 197 becomes cysteine (A197C), at the edge of the cleft 3. **3 · Attach a dye** The dye MDCC is linked to that cysteine; open, it glows weakly 4. **4 · Phosphate closes it** The lobes clamp shut and the dye glows about 5 times brighter **Designing the sensor.** Because the dye sits where the lobes move, closing the protein changes the dye’s surroundings and makes it brighter. Phosphate binds tightly (dissociation constant about 0.1 µM) and fast, so the signal follows phosphate release in real time (Brune et al. 1994). Schematic, drawn for this site. ## From light to kinetics Each GTP that FtsZ splits releases one phosphate, so the sensor’s brightness rises as FtsZ works. A calibration with known amounts of phosphate turns brightness into concentration, and the slope over time gives the rate. Because FtsZ hydrolyses GTP mainly once it has assembled into filaments, almost nothing happens below a critical concentration of FtsZ (Romberg & Levin 2003). (evidence: established) Above it, rates measured at several GTP concentrations give the Michaelis–Menten parameters Vmax and Km. *[Here the web page has an interactive activity; open the source URL to use it.]* ## What I did - I prepared the sensor: mutagenesis, expression, purification, labelling and quality checks. - I calibrated how its fluorescence changes with phosphate. - I used it to follow GTP use by **FtsZ from *Spiroplasma melliferum*** (a cell-wall-less bacterium studied in the lab) under different conditions, and wrote a step-by-step analysis pipeline from raw fluorescence to Michaelis–Menten parameters. ## What I took away - **Good measurement is a design problem:** contamination (phosphate is everywhere!), calibration ranges and blanks decide what you can trust. - **Real enzymes are richer than textbooks.** Because FtsZ only uses GTP once it polymerises, it does not obey the simplest enzyme model, and that is exactly what makes it interesting. ## Why it matters New antibiotics need new targets. FtsZ inhibitors have reached early clinical testing, and none has yet passed Phase 2. Sensitive, real-time assays like this one are how candidate molecules get screened. ## Methods used - Site-directed mutagenesis and cloning - Protein expression and purification (affinity and ion-exchange chromatography) - Fluorescent dye labelling - Fluorescence and absorbance spectroscopy - Enzyme kinetics (Michaelis–Menten) - Data analysis pipelines [What these methods are →](https://khushisingh.in/research/toolbox/) ## References 1. Brune M, Hunter JL, Corrie JET, Webb MR (1994) Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33:8262–8271. [doi:10.1021/bi00193a013](https://doi.org/10.1021/bi00193a013) 2. Solscheid C, et al. (2015) Development of a reagentless biosensor for inorganic phosphate, applicable over a wide concentration range. Biochemistry 54:5054–5062. [doi:10.1021/acs.biochem.5b00449](https://doi.org/10.1021/acs.biochem.5b00449) 3. Chakraborty J, et al. (2024) Dynamics of interdomain rotation facilitates FtsZ filament assembly. Journal of Biological Chemistry 300:107336. [doi:10.1016/j.jbc.2024.107336](https://doi.org/10.1016/j.jbc.2024.107336) 4. Brune M, et al. (1998) Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37:10370–10380. [doi:10.1021/bi9804277](https://doi.org/10.1021/bi9804277) 5. Löwe J, Amos LA (1998) Crystal structure of the bacterial cell-division protein FtsZ. Nature 391:203–206. [doi:10.1038/34472](https://doi.org/10.1038/34472) 6. Mukherjee A, Lutkenhaus J (1998) Dynamic assembly of FtsZ regulated by GTP hydrolysis. EMBO Journal 17:462–469. [doi:10.1093/emboj/17.2.462](https://doi.org/10.1093/emboj/17.2.462) 7. Romberg L, Levin PA (2003) Assembly dynamics of the bacterial cell division protein FtsZ: poised at the edge of stability. Annual Review of Microbiology 57:125–154. [doi:10.1146/annurev.micro.57.012903.074300](https://doi.org/10.1146/annurev.micro.57.012903.074300) 8. Erickson HP, Anderson DE, Osawa M (2010) FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiology and Molecular Biology Reviews 74:504–528. [doi:10.1128/mmbr.00021-10](https://doi.org/10.1128/mmbr.00021-10) 9. Haydon DJ, et al. (2008) An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 321:1673–1675. [doi:10.1126/science.1159961](https://doi.org/10.1126/science.1159961)