Characterization of the Assembly Dynamics of Streptococcus Pneumoniae FtsZ using Intrinsic Tryptophan Fluorescence

Abstract

FtsZ is a prokaryotic cytoskeletal protein, which assembles into the Z‐ring at the division site and orchestrates the cell division process in bacteria. FtsZ has GTPase activity and self‐assembles to form polymers. We investigated the assembly characteristics of FtsZ from the pathogenic organism, Streptococcus pneumoniae. The critical concentration for FtsZ polymerization was found to be 2.4 ± 0.1 and 2.2 ± 0.2 μM measured using sedimentation assay and GTPase assay, respectively. The results indicated that the GTPase activity and assembly are closely linked to each other. We identified that FtsZ from Streptococcus species contains two native tryptophan (Trp) residues unique to this genus. Interestingly, we observed that during polymerization, the tryptophan fluorescence of FtsZ increases showing that the assembly of FtsZ can be monitored using the native tryptophan fluorescence. Using tryptophan fluorescence along with light scattering, sedimentation assay and electron microscopy we examined the effects of divalent cations, calcium and magnesium, on FtsZ assembly. Divalent magnesium enhanced the tryptophan fluorescence change during FtsZ assembly showing that it promoted the longitudinal assembly of FtsZ. Divalent calcium induced the bundling of FtsZ filaments thereby affecting the lateral interactions of FtsZ polymers but did not enhance the tryptophan fluorescence, showing the sensitivity of tryptophan fluorescence to longitudinal assembly. Next, we sought to investigate the contribution of individual tryptophan residues to fluorescence of FtsZ and use them for understanding the assembly of FtsZ. A homology model for FtsZ from Streptococcus pneumoniae showed that the two tryptophan residues were present at the C‐terminal of the protein – one at the 298th position of FtsZ near the T7 loop and the other at 378th position in the C‐terminal tail region. We used site‐directed mutagenesis to introduce point mutations to FtsZ at these positions to mutate individual tryptophan residues to cysteine or phenylalanine residues to create W294C/F and W378C/F mutant proteins. W378C/F mutants showed a similar change in tryptophan fluorescence during polymerization as of wild‐type, while W294C/F mutants did not show any change. This indicated that the Trp at the 294th position is responsible for the change in fluorescence during polymerization. This further suggested that the environment around 294W changes during the polymerization while the local environment around the 378W does not change. The two tryptophan residues in the protein allow us to use the intrinsic tryptophan fluorescence to gain new insights into FtsZ assembly. Further, it would be interesting to explore the use of these mutant proteins in following the domain movements and conformational changes occurring during the assembly and disassembly of FtsZ.Support or Funding InformationThe work is supported by a grant from Department of Science and Technology (DST, India) to D.P.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.