Cell-free protein synthesis of membrane (1,3)-beta-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.Agalya Periasamy, Nadim Shadiac, Amritha Amalraj, Soňa Garajová, Yagnesh Nagarajan, Shane Waters, Haydyn D.T. Mertens, Maria Hrmov

Investigation of the mitochondrial translocase of the outer membrane (TOM) of Drosophila melanogaster

© 2018 Dr. Agalya PeriasamyThe macromolecular protein translocation machinery of the outer mitochondrial membrane, TOM, mediates the import of nuclear-encoded mitochondria-bound precursor proteins. Moreover, a demonstrated role in importing pathogenic polypeptides signifies its medical relevance as a potential therapeutic target. Structural investigations of TOM have to date been limited to lower eukaryotic fungi from which the components can be purified in reasonable quantities. The underlying objective of this thesis is to seek and interrogate the structure of a metazoan TOM complex in order to unravel features of greater relevance to the human TOM complex. To this end, the fruit fly Drosophila melanogaster was evaluated as an in vivo system for transgenic expression of the translocase components for production of purified TOM amenable to structural analysis by cryo-EM. The capacity for targeted, tuneable expression in specific tissues and developmental stages and a provision for phenotypic readouts in Drosophila flies holds particular advantages over cell culture systems for investigation of structure-function and wider interactions of TOM in the context of human biology and disease. Chapter 1 offers an introduction to the mitochondrial import system with particular focus on the organisation of TOM, functional and structural attributes of subunits of the translocase and reported involvement in human diseases. Chapter 2 describes the homologous expression of epitope-tagged Tom40 and Tom22 in Drosophila followed by an account of the characterization and successful isolation of the Drosophila TOM assembly suitable for single particle EM analysis. Tryptic-digest mass spectrometric analysis of purified TOM, has identified the potential Drosophila orthologues of Tom5 and Tom6 and, point to novel associations of TOM with other mitochondrial proteins namely, VDAC and ANT. Chapter 3 presents preliminary cryo-EM data of purified Drosophila TOM. 2D class averaging has revealed the presence of three-pore particles, albeit, certain detergent related issues remain to be addressed. Further sample optimization strategies for prospective cryo-EM studies are discussed. Chapter 4 details an investigation of a defective eye phenotype that manifests in the Drosophila eye as a consequence of elevated Tom40 expression. Analysis of active caspase levels has demonstrated a cell death basis for the observed phenotype. Lastly, in Chapter 5, results and findings of the thesis are discussed in the context of reported biochemical and structural data, with emphasis on the organisation and stoichiometry of TOM. A hypothetical model that likely explains the interaction of VDAC with TOM is presented followed by some concluding remarks on the significance and future directions of the project