| Author: | Gong, Furong |
| Title: | A study of the interaction of periplasmic FtsQ, FtsB and FtsL of Escherichia coli by mass spectrometry |
| Advisors: | Wong, K. Y. (ABCT) |
| Degree: | M.Phil. |
| Year: | 2021 |
| Subject: | Cell division Cell proliferation Escherichia coli -- Genetics Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 238 pages : color illustrations |
| Language: | English |
| Abstract: | Cell division is an essential process for bacterial proliferation that is mainly controlled and progressed by a protein complex called 'divisome'. Divisome is complicated machinery mediating the cell division process through the regulation in the synthesis and hydrolysis of the cell wall peptidoglycan, as well as the invagination of the cell membrane. Recent studies of the E. coli divisome showed that at least ten proteins are essential for the proper functioning of a mature divisome unit. These essential proteins are recruited to the division site following a hierarchical order: FtsZ > FtsA/ZipA > FtsK > FtsQ > FtsB/FtsL > FtsW > FtsI > FtsN. The arrival of the latest protein FtsN triggers the activation of peptidoglycan synthesis for the lateral growth of the cell wall. Eventually, a mother bacterial cell is divided into two daughter cells by the construction and invagination of the cell wall and membrane in a well-controlled manner. FtsQBL, as the periplasmic connector, plays an important role in the cell division process. FtsQ, FtsB, and FtsL form a protein complex that acts as a key to control the synthesis of cell wall peptidoglycan before the divisome assembly. The FtsQBL complex is believed to have two conformations to complete the task, including an 'off' state for suppression and an 'on' state for stimulation. The structures and properties of periplasmic FtsQ, FtsB, and FtsL have been studied with efforts in the past decade. The crystal structure of the entire FtsQ periplasmic domain and a small FtsB periplasmic segment have been reported at the end of 2018. However, the entire structure and interaction sites of the FtsQBL complex remain unclear. To obtain a better FtsQBL model, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied in this project to study the difference in solvent accessibilities of the periplasmic domains of FtsQ (FtsQp), FtsB (FtsBp), and FtsL (FtsLp) before and after the complexation. The HDX-MS results could indicate the protein segments that are potentially involved in the FtsQp-FtsBp-FtsLp interaction. Plasmids encoding genetic sequences of FtsQp, FtsBp, FtsLp, FtsQpBp, FtsBpLp, and FtsQpBpLp, were reconstructed for the study. These target proteins and complexes were then overexpressed and isolated by Ni-NTA affinity chromatography, and their masses were confirmed by SDS-PAGE and electrospray mass spectrometry. The native nano-electrospray mass spectrometric analysis suggested that the individual Fts-proteins were mainly retained in their monomeric states, whereas the co-expressed FtsQp, FtsBp, and FtsLp proteins preferred to form trimeric complexes in a 1:1:1 manner. Moreover, the HDX-MS data indicates that FtsQp predominantly interacts with FtsBp in the FtsQpBpLp-bound state. The H5 helix and the S11 and S12 β-sheets in the C-terminal FtsQp (residues between Glu225 and Glu263), as well as the C-S1 β-sheet and C-H1 helix within the FtsBp Glu69 - Phe84 subdomain, are involved in the FtsQp-FtsBp binding as shown by their decreased deuterium incorporation level after the FtsQpBp and FtsQpBpLp complexations. The presence of FtsQp may induce additional interaction between FtsBp and FtsLp. FtsBp in the FtsQpBpLp-bound state may interact with FtsLp (residues between Glu89 and Glu103) via the N-H1 helix (residues Ala37 to Leu60). However, this interaction was not detected in FtsBp and FtsLp upon the FtsBpLp complex formation, suggesting that the FtsBp-FtsLp oligomerization may mainly be triggered by the charge interactions between e5-and k5-loops. In addition, unfolding in the N-terminal FtsLp was detected during the analysis, as revealed by the significant increase in deuterium uptake within the region (residues Leu63 to Glu87) after the formation of the FtsBpLp or FtsQpBpLp complexes. The HDX-MS results help better understand the structure, binding interface, and binding mode of the FtsQpBpLp complex. |
| Rights: | All rights reserved |
| Access: | open access |
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