Author: Daniel Carrillo Baez
Carrillo Baez, Daniel, 2023 DECIPHERING THE TOPOLOGY OF THE E. COLI INNER MEMBRANE PROTEOME , Flinders University, College of Medicine and Public Health
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Inner membrane proteins (IMPs) are essential components of the gram-negative bacteria inner cell membrane. They perform various functions, including nutrient uptake, energy metabolism, signal transduction, stress responses, and cell division. The number of IMPs in E. coli is estimated to be around 900, and collectively, they are involved in various cellular processes essential for the bacteria's survival and growth. Understanding the orientation of IMPs, i.e., whether functional domains are exposed to the cytoplasm or periplasm, is essential for elucidating their functions and the molecular mechanism of interaction with other cellular components. Thus, this thesis aims to provide a novel methodology based on dynamic location measurements that can reveal the topology of the E. coli IMPs.
Specifically, the current experimental topology mapping methods were expanded by (i) using a pluripotent fluorescent protein capable of expressing in the cytoplasm and periplasm and (ii) step changes in pH. sfGFP was utilized in an innovative, dynamic measurement approach, introducing pH step changes to induce variation in fluorescence intensity. These changes reported on the intracellular versus periplasmic location of sfGFP, as only in the latter scenario leads to a sustained change in fluorescence (Wilks and Slonczewski, 2007). The following steps were meticulously planned and carried out to achieve this goal. First, a new plasmid capable of expressing sfGFP was developed. Second, the replication capabilities were improved to acceptable levels. Third, a systematic exploration of HCl volume and cell numbers was developed to find the optimal conditions. Fourth, clear data measuring parameters were defined to ensure precise data analysis. Lastly, the proposed method was tested in eight test proteins with diverse topologies and lengths, similar to those found in all IMPs. This found that the method successfully predicts the orientation of the C-terminus by clearly showing a difference in fluorescence recovery between sfGFP localized in the cytoplasm and periplasm. The results of this thesis indicate that a novel method to map membrane protein topology was successfully developed. Further work will focus on the application of this method to the entire inner proteome and correlating the obtained results to existing experimental and computational models. This method may also serve as a proxy for detecting membrane protein insertion and folding in the context of various conditions.
Keywords: Inner membrane protein, E. coli, Membrane protein, Superfolder green fluorescent protein, pH change, Fluorescence intensity, Localization, C-terminus, N-terminus
Subject: Medical Biotechnology thesis
Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Professor Harald Janovjak