| Author: | Zheng, Yuanyuan |
| Title: | A mass spectrometric study of the conformational change of Escherichia coli lipid II flippase MurJ |
| Advisors: | Wong, Kwok-yin (ABCT) |
| Degree: | Ph.D. |
| Year: | 2025 |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xi, 161 pages : color illustrations |
| Language: | English |
| Abstract: | MurJ is a membrane protein that is responsible for the transport of peptidoglycan precursor lipid II to the outside of the membrane during bacterial cell wall synthesis in E. coli. Previous studies have characterized the structure of MurJ in different conformations, identified the potential binding site of lipid II, and hypothesized that MurJ transports its substrate lipid II by alternating its structure between the inward and outward conformations. However, the molecular mechanism of lipid II translocation is largely unknown. For instance, the driving force involved in the conformational alternation and the dynamic changes between these conformations are still unclear. In this study, three recombinant MurJ proteins from E. coli were expressed and purified. The protein structure was analyzed by circular dichroism (CD) spectroscopy. The results indicated that the helical secondary structure of the MurJ protein was maintained despite variations in pH over the range of 1.0-10.0 and exposure to high concentrations (6 M) of the denaturing agent Guanidine-HCl. This observation highlights the stability of protein secondary structures in response to the environment. To elucidate the driving force in structural transition of MurJ, a combination of structure-guided cysteine cross-linking and protein native mass spectrometry was employed to detect alternations in protein conformation in vivo. Two double mutant protein models were constructed, which could fix the protein in one preferred conformation after cross-linking, namely V43C_T251C (inward-open) and E70C_A296C (outward-open). By adding compounds that interfere with the cell membrane potential, it was found that the disruption of the proton motive force by the compound 3,3',4',5-Tetrachlorosalicylanilide (TCS) significantly hindered the ability of mutant E70C_A296C to transition into the outward-facing configuration required for successful cross-linking. However, the mutant V43C_T251C was able to achieve in vivo cross-linking in the inward configuration under this condition. This indicates that the proton motive force is essential for the conformational transition of MurJ. The dynamic movement of MurJ protein in two different conformations (inward-open and outward-open) in solution was studied by using hydrogen-deuterium-exchange mass spectrometry (HDX-MS). Comparative analysis of deuterium uptake in the two conformations at different timepoints showed that amino acid residues located in the central cavity of the protein displayed little variation in deuterium uptake differences. This region corresponds to the binding site of the lipid II headgroup. On the other hand, amino acid residues located in transmembrane helices TM1, 2, 7 and 9 showed significant change (> 0.5 Da) in deuterium uptake, indicating these peptides are more flexible in nature. This suggests that MurJ may facilitate the substrate flipping process by maintaining its interaction with the lipid II headgroup while changing the binding with lipid tail. Thus, the protein structure located within the central cavity exhibits relative rigidity, while the region adjacent to the lipid tails exhibits greater flexibility. |
| Rights: | All rights reserved |
| Access: | open access |
Copyright Undertaking
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item:
https://theses.lib.polyu.edu.hk/handle/200/13760