- PhD, University of Oxford
- Assistant Professor, Molecular Physiology and Biological Physics
Transport of biopolymers across biological membranes with a particular interest in polysaccharide and protein translocation.
Many biopolymers, such as polypeptides, nucleic acids and polysaccharides, have to cross at least one membrane to reach their final destination. Our knowledge has expended significantly on the translocation and membrane integration of polypeptides; however, very little is known about how one of the most hydrophilic polymers, the polysaccharide, can cross the hydrophobic barrier surrounding every cell.
Polysaccharides come in many forms and serve a multitude of different functions. For example, starch and glycogen are means of energy storage, cellulose and chitin confer structural stability to the cell and hyaluronan influences cell differentiation, proliferation and migration. To serve their ultimate purpose, many of these polysaccharides, which can be several microns in length, are transported across biological bilayers. In many cases membrane embedded, processive polysaccharide synthases link intracellular building blocks (activated sugars) and translocate the growing polysaccharide chain across the bilayer. This process requires (1) the specific binding of the activated sugars by intracellular domains, (2) stereospecific glycosyltransferase activities to covalently link the building blocks, (3) the formation of a channel spanning the bilayer and (4) the translocation of the growing polysaccharide chain across the membrane. Mechanistically, this process promises to be fundamentally different from protein translocation, since the synthase that generates the polymer is combined with the channel across the membrane in a single polypeptide chain.
Studying the translocation of polysaccharides across biological membranes necessitates a broad spectrum of techniques. Initially, target proteins will be cloned and expressed followed by purification. With high quality material in hand, biophysical and biochemical studies will follow. A major aim of the laboratory is the structural characterization of glycosyltransferases involved in hyaluronan, cellulose and curdlan synthesis. Simultaneously, we will work to establish an in vitro polysaccharide translocation assay by reconstituting purified enzymes into proteoliposomes. This will allow us to (1) reproduce the biosynthesis and translocation of the polymer outside the cell, (2) to develop assays that identify the path of the polysaccharide through the membrane channel and (3) to identify conditions that will trap translocation intermediates.
As an interdisciplinary approach, we will use electron microscopy to study the supra-molecular assemblies some of the glycosyltransferases form in native membranes as well as cell biology techniques to study the surface expression of matrix polysaccharides in a physiological context.
Techniques to be applied:
Recombinant DNA-, Molecular Biology techniques
Protein expression in bacterial and eukaryotic systems
Reconstitution of proteins into lipid vesicles
Enzyme activity assays
Protein purification (metal affinity-, ion exchange- and gel filtration chromatography)
- Hubbard C, McNamara J, Azumaya C, Patel M, Zimmer J. The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan. Journal of molecular biology. 2012;418(1): 21-31. PMID: 22343360
- Mazur O, Zimmer J. Apo- and cellopentaose-bound structures of the bacterial cellulose synthase subunit BcsZ. The Journal of biological chemistry. 2011;286(20): 17601-6. PMID: 21454578 | PMCID: PMC3093835
- Morgan J. L., Strumillo J., Zimmer J.. Crystallographic snapshot of cellulose synthesis and membrane translocation Nature. 493(7431): 181-6.
- Omadjela O., Narahari A., Strumillo J., Melida H., Mazur O., Bulone V., Zimmer J.. BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis Proc Natl Acad Sci U S A. 110(44): 17856-61.