Scientific publications
science Portfolio
5th June, 2023

Published work: Activator-induced conformational changes regulate division-associated peptidoglycan amidases
Abstract
AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.
Skills shown
This published research paper was the result of a collaborative effort among members of multiple interdisciplinary research groups, combining expertise in biochemistry, X-ray crystallography, bioinformatics, data analysis, microbiology, genetics, and protein modeling. By leveraging the diverse strengths, skills, and experiences of each team member, the project achieved a successful outcome. The research was meticulously conducted, thoroughly reviewed, and ultimately published in a reputable journal, reflecting the collective contributions and dedication of the team.
As part of my research, I gained extensive hands-on experience with a wide range of laboratory techniques. These include maintaining high standards of health and safety in the lab, mastering aseptic techniques to prevent contamination, and performing bacterial genetic manipulation. I also developed skills in large-scale bacterial culturing, cloning, polymerase chain reaction (PCR), and complementation experiments. Additionally, I conducted microscopy for detailed analysis, protein expression and purification to study molecular interactions, and gel electrophoresis for DNA, RNA, and protein analysis. My expertise also extends to performing minimum inhibitory concentration (MIC) experiments to evaluate antibiotic efficacy, showcasing a comprehensive skill set in molecular biology and microbiology.
Effective communication is a fundamental aspect of being a successful research scientist and is crucial for fostering a collaborative and productive work environment. This involves presenting complex data and experimental results to a wide range of audiences, from experts in the field to non-specialists. It also requires a deep understanding of related topics to provide context and clarity when discussing findings. Additionally, empathy plays a vital role in recognizing and appreciating the strengths and weaknesses of fellow scientists, which helps build a supportive and collaborative team dynamic. Strong communication ensures that ideas are shared clearly, feedback is constructive, and collective goals are achieved.
In addition to practical laboratory techniques, data analysis is an essential skill for any research scientist. Proficiency in tools like Microsoft Excel is crucial for data visualization and statistical analysis, allowing scientists to effectively interpret and present complex datasets. Specifically in biology, bioinformatics plays a significant role in tasks such as genetic sequence alignments, protein and ligand modeling, and the development of predictive models. For this research paper, large volumes of growth curve data were experimentally generated, meticulously analyzed in Excel, and then presented using GraphPad Prism to highlight key trends and results. This combination of data analysis tools ensured accurate interpretation and effective communication of the findings.
For all research publications, it is essential to critically analyze the work to ensure its robustness and reliability. This involves asking key questions such as: Are additional experiments needed to support or prove the hypothesis? Are there any controls that should be implemented to validate the data and ensure its accuracy? Furthermore, it is important to evaluate whether the methods used are reliable and capable of producing consistent results. A critical review of these aspects ensures that the research findings are well-founded, reproducible, and contribute meaningfully to the broader scientific community.
Due to the diverse range of challenges, techniques, and scientific theories encountered in research, adaptability is essential. Being able to adjust to the specific requirements of each project and effectively problem-solve issues related to data or experimental methods is a common aspect of scientific work. The ability to remain flexible with approaches and methods, while striving to identify the most efficient, effective, and reproducible data generation strategies, is crucial for a research scientist. This flexibility was particularly important for the success of this paper, ensuring that the research was both robust and capable of producing reliable results.
Link
Full paper: https://www.pnas.org/doi/10.1073/pnas.2302580120
1st April, 2023

Ph.D Thesis: Investigation of the structural
regulation of periplasmic
amidases during cell division in
Escherichia coli
Abstract
During bacterial cell division, amidases hydrolyse peptidoglycan to allow daughter cell separation. During the final stages of cell division, septal peptidoglycan between the two daughter cells prevents cell separation. Proteins called Nacetylmuramoyl-L-alanine type 3 amidases hydrolyse the amide bond between glycan strands. This allows successful daughter cell separation. This hydrolase activity is tightly regulated by a pathway of protein interactions to avoid uncontrolled peptidoglycan degradation. The exact structural mechanism of the protein interaction pathway involved in regulating the activity of such amidases is currently unknown. Here it is shown, the molecular regulatory mechanism by which, Escherichia coli amidase, AmiA, is activated by the interaction of EnvC. It was found that EnvC, an amidase regulator, is self-regulated by a restraining helix over the amidase binding LytM domain evidenced by bacterial-two-hybrid and co-purification analysis of EnvC truncated variants. By solving a 2.35 angstrom structure of E. coli and building an AmiA bound to EnvC LytM domain model, the molecular mechanism of the removal of a blocking helix over AmiA’s active site by the interaction of an interaction helix in AmiA to EnvC. These results show a stringently structurally regulated amidase activation pathway, with no availability of the amidase active site without interaction with a regulator protein. This is a potentially conserved regulatory system that could be use to model amidase regulation in other bacterial species. As a part of fundamental biology in a model bacterial system, it is anticipated that the understanding found in this research could extend to understanding related cell division pathways in other bacterial species. As an application of this regulation mechanism, there is a potential antibiotic target to be found in cell division amidases, as without self-regulation, amidases may uncontrollably hydrolyse peptidoglycan, weakening the integrity of cells to make them more susceptible to other antibiotic treatments.
Skills shown
As with all research Ph.D. projects, careful planning, determination, and resilience are fundamental to success. This experience has significantly strengthened my ability to work independently, fostering a strong sense of autonomy in managing my research. At the same time, it has underscored the importance of collaboration and networking, allowing me to exchange ideas and gain valuable insights from fellow academics. By balancing independent work with collaborative efforts, I have been able to enhance my research and develop a deeper understanding of my field.
As part of my research, I gained experience with a wide range of laboratory techniques, including health and safety protocols, aseptic techniques, bacterial genetic manipulation, large-scale bacterial culturing, cloning, polymerase chain reaction (PCR), complementation experiments, microscopy, protein expression and purification, gel electrophoresis, and minimum inhibitory concentration (MIC) experiments for antibiotics.
Effective communication is a critical skill for any research scientist, playing a central role in fostering a collaborative and productive work environment. It involves presenting complex data and experimental findings to a variety of audiences, from specialists to non-experts, in a clear and accessible way. In addition to communicating results, a strong understanding of related topics is necessary to provide context and facilitate informed discussions. Equally important is demonstrating empathy for colleagues, recognizing their strengths and weaknesses, and fostering an atmosphere of mutual respect. This ability to engage effectively with others not only enhances the quality of the work but also promotes a positive, supportive research environment.
As part of my Ph.D. program, I received comprehensive training in statistical theory and data analysis using Microsoft Excel and R, equipping me with the skills to manage and interpret complex datasets effectively. In the field of biochemistry, I developed expertise in specialized areas such as bioinformatics, genetic sequencing data analysis, and protein structure prediction and evaluation. These skills have enabled me to integrate computational and experimental approaches to address research questions with precision and depth.
Scientific research involves constant refinement of hypotheses as new data emerges, along with addressing errors in experiments. This process demands a deep understanding of the work to identify and resolve potential issues. Problem-solving in science also fosters creativity, encouraging innovative solutions that others may not have considered or prompting collaboration with those who have encountered and resolved similar challenges.
Research often operates within financial constraints or set time-frames, making effective time management essential. This involves prioritizing promising research paths and running tasks in parallel to maximize efficiency. Strong planning skills are crucial, including anticipating potential problems, allocating time for adjustments, and ensuring a thorough understanding of the subject area.
Link
23rd October, 2020

Published work: Insights into bacterial cell division from a structure of EnvC bound to the FtsX periplasmic domain
Abstract
FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In Escherichia coli, FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a “restraining arm” suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which E. coli would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases.
Skills shown
This published research paper was the result of a collaborative effort among members of multiple interdisciplinary research groups, combining expertise in biochemistry, X-ray crystallography, bioinformatics, data analysis, microbiology, genetics, and protein modeling. By leveraging the diverse strengths, skills, and experiences of each team member, the project achieved a successful outcome. The research was meticulously conducted, thoroughly reviewed, and ultimately published in a reputable journal, reflecting the collective contributions and dedication of the team.
As part of my research, I gained extensive hands-on experience with a wide range of laboratory techniques. These include maintaining high standards of health and safety in the lab, mastering aseptic techniques to prevent contamination, and performing bacterial genetic manipulation. I also developed skills in large-scale bacterial culturing, cloning, polymerase chain reaction (PCR), and complementation experiments. Additionally, I conducted microscopy for detailed analysis, protein expression and purification to study molecular interactions, and gel electrophoresis for DNA, RNA, and protein analysis. My expertise also extends to performing minimum inhibitory concentration (MIC) experiments to evaluate antibiotic efficacy, showcasing a comprehensive skill set in molecular biology and microbiology.
Effective communication is a fundamental aspect of being a successful research scientist and is crucial for fostering a collaborative and productive work environment. This involves presenting complex data and experimental results to a wide range of audiences, from experts in the field to non-specialists. It also requires a deep understanding of related topics to provide context and clarity when discussing findings. Additionally, empathy plays a vital role in recognizing and appreciating the strengths and weaknesses of fellow scientists, which helps build a supportive and collaborative team dynamic. Strong communication ensures that ideas are shared clearly, feedback is constructive, and collective goals are achieved.
In addition to practical laboratory techniques, data analysis is an essential skill for any research scientist. Proficiency in tools like Microsoft Excel is crucial for data visualization and statistical analysis, allowing scientists to effectively interpret and present complex datasets. Specifically in biology, bioinformatics plays a significant role in tasks such as genetic sequence alignments, protein and ligand modeling, and the development of predictive models. For this research paper, large volumes of growth curve data were experimentally generated, meticulously analyzed in Excel, and then presented using GraphPad Prism to highlight key trends and results. This combination of data analysis tools ensured accurate interpretation and effective communication of the findings.
For all research publications, it is essential to critically analyze the work to ensure its robustness and reliability. This involves asking key questions such as: Are additional experiments needed to support or prove the hypothesis? Are there any controls that should be implemented to validate the data and ensure its accuracy? Furthermore, it is important to evaluate whether the methods used are reliable and capable of producing consistent results. A critical review of these aspects ensures that the research findings are well-founded, reproducible, and contribute meaningfully to the broader scientific community.
Due to the diverse range of challenges, techniques, and scientific theories encountered in research, adaptability is essential. Being able to adjust to the specific requirements of each project and effectively problem-solve issues related to data or experimental methods is a common aspect of scientific work. The ability to remain flexible with approaches and methods, while striving to identify the most efficient, effective, and reproducible data generation strategies, is crucial for a research scientist. This flexibility was particularly important for the success of this paper, ensuring that the research was both robust and capable of producing reliable results.
Link
Full paper: https://www.pnas.org/doi/10.1073/pnas.2017134117
23rd October, 2020

Masters Thesis: The characterisation of Eukaryotic like serine/threonine kinases
involved in antibiotic resistance in
Gram Positive bacteria
Abstract
Gram-positive bacteria are becoming the major cause of multi-drug resistant bacterial infections. These include nosocomial infections from vancomycin resistant Enterococcus faecalis or the opportunistic colonisation of the bowel by Clostridium difficile post broad spectrum antibiotic treatments. Gram-positive bacteria have developed their own intrinsic resistances to antibiotics along with an enhanced range of mechanisms acquired from other resistant bacterial strains. Bacteria use transmembrane signalling systems to regulate a range of cellular processes including virulence, growth and antibiotic resistance. One such system, originally only thought to be present in Eukaryotes, Eukaryotic-like Serine/Threonine kinases (eSTKs) and their cognate phosphatases are known to regulate such cellular processes. These systems contain a cytosolic kinase domain which autophosphorylate upon detection of an extracellular signal using extracellular PASTA domains. In Enterococcus faecalis, there is an example of this system called IreK/IreP, but also similar systems are found in Enterococcus faecium and Clostridium difficile.
This project was a preliminary study into characterising the autophosphorylation activity of eSTK kinase domains from E. faecalis, E. faecium and C. difficile. Using phosphate detection methods, this work discovered eSTK kinase domains are phosphorylated during overexpression in E. coli and can be successfully dephosphorylated by general and cognate phosphatases. This led to the investigation of the effect of an ATP-competitive kinase inhibitor staurosporine on kinase function in vivo and in vitro. The inhibitor was found to reduce the autophosphorylation activity of IreK and bind with high affinity to the ADP binding site of the kinase domain. During in vivo experiments, Staurosporine was discovered to reduce Cephalosporin resistance in C. difficile by over 100-fold. The project contributes to the growing research field of bacterial signalling systems and the prospect of using kinase inhibitors as a combinational therapy against multi-drug resistant bacteria.
Skills shown
As with all research projects, careful planning, determination, and resilience are fundamental to success. This experience has significantly strengthened my ability to work independently, fostering a strong sense of autonomy in managing my research. At the same time, it has underscored the importance of collaboration and networking, allowing me to exchange ideas and gain valuable insights from fellow academics. By balancing independent work with collaborative efforts, I have been able to enhance my research and develop a deeper understanding of my field.
As part of my research, I gained experience with a wide range of laboratory techniques, including health and safety protocols, aseptic techniques, bacterial genetic manipulation, large-scale bacterial culturing, cloning, polymerase chain reaction (PCR), complementation experiments, microscopy, protein expression and purification, gel electrophoresis, and minimum inhibitory concentration (MIC) experiments for antibiotics.
Effective communication is a critical skill for any research scientist, playing a central role in fostering a collaborative and productive work environment. It involves presenting complex data and experimental findings to a variety of audiences, from specialists to non-experts, in a clear and accessible way. In addition to communicating results, a strong understanding of related topics is necessary to provide context and facilitate informed discussions. Equally important is demonstrating empathy for colleagues, recognizing their strengths and weaknesses, and fostering an atmosphere of mutual respect. This ability to engage effectively with others not only enhances the quality of the work but also promotes a positive, supportive research environment.
As part of my Ph.D. program, I received comprehensive training in statistical theory and data analysis using Microsoft Excel and R, equipping me with the skills to manage and interpret complex datasets effectively. In the field of biochemistry, I developed expertise in specialized areas such as bioinformatics, genetic sequencing data analysis, and protein structure prediction and evaluation. These skills have enabled me to integrate computational and experimental approaches to address research questions with precision and depth.
Scientific research involves constant refinement of hypotheses as new data emerges, along with addressing errors in experiments. This process demands a deep understanding of the work to identify and resolve potential issues. Problem-solving in science also fosters creativity, encouraging innovative solutions that others may not have considered or prompting collaboration with those who have encountered and resolved similar challenges.
Research often operates within financial constraints or set time-frames, making effective time management essential. This involves prioritizing promising research paths and running tasks in parallel to maximize efficiency. Strong planning skills are crucial, including anticipating potential problems, allocating time for adjustments, and ensuring a thorough understanding of the subject area.