Structural and functional characterization of membrane proteins involved in the peptidoglycan synthesis

Author(s)

Lucas Mayrink Assis

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-07-26

Examining board members

Andréa Dessen; Aline Mara dos Santos; Andre Luís Berteli Ambrosio; Juliana Helena Costa Smetana; Juliana Ferreira de Oliveira

• Advisor: Andréa Dessen

Abstract

The cell wall plays an essential role in the maintenance of bacterial survival, preventing osmotic lysis and providing rigidity and shape to the cell. The enzymes involved in the biosynthesis of its central component, the peptidoglycan, are potential targets for novel pharmaceuticals. In addition, most of this metabolic pathway is well-characterized structurally and functionally. One of the exceptions consists of the translocation step of the precursor substrate named lipid II. The proteins that mediate this cellular role are designated, generically, as flippases. In this sense, the objective of this work was the structural and functional characterization of the RodA protein, a membrane protein that participates in the process of lipid II translocation during the cell elongation, being part of the multi-protein complex called the elongasome. Additionally, this study also addressed the structural understanding of the chimeric proteins (Flippase-PBPs), which contain the enzymatic functions of both flippase and Penicillin Binding Protein (PBP). Most of the doctoral work described here was performed at the Brazilian Biosciences National Laboratory (LNBio), Campinas, Brazil; nevertheless, one part of the study was carried out at the Institut de Biologie Structurale (IBS), Grenoble, France. In terms of the methodologies, our approach consisted in protein expression using bacterial- and cell-free-based systems, purification and selection of detergents suitable for the solubilization of the membrane proteins, hydrodynamic characterization through analytical ultracentrifugation and dynamic light scattering, crystallization (in surfo and in cubo), and characterization of the enzymatic activity concerning the flippase and GTase activities of RodA. The results thus obtained showed the efficiency of the cell-free system in expressing the proteins and, additionally, showed that this type of system leads to simpler purification protocols when compared to protocols developed with bacterial expression systems. The characterization of the Flippase-PBP-detergent complex demonstrated that it is monomeric and numerically estimated the values for its hydrodynamic radius and its frictional quotient. The crystallization in cubo was implemented as the main crystallization technique and initial trials were performed with RodA and Flippase-PBP. The results of the functional characterization of RodA (E. coli and B. subtilis) will be published shortly and demonstrate the enzymatic action of this protein with regard to the translocation of lipid II and the catalytic action of transglycosylation (AU)

• FAPESP's process: 13/22681-0 - Structural characterization of membrane protein complexes involved in bacterial cell wall biosynthesis
• Grantee: Lucas Mayrink Assis
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)