Student projects - Eefjan Breukink
Plectasin is a antimicrobial peptide (AMP) first discovered back in 2005 in the fungus Pseudoplectania nigrella. Plectasin and its variants (e.g. NZ2114, MP1102) have excellent bactericidal activity against a variety of gram positive bacteria. This includes some clinically relevant strains, among which MRSA, infamous in hospitals for causing hard to treat infections. Lipid-II binding is thought to be the mechanism of action of these peptides but much remains unclear about the structural details.
Unfortunately, studying the atomic structure of peptide-Lipid II complexes in their native environment, a membrane, by conventional techniques -such as X-ray diffraction- is challenging. Furthermore, without these data the development of these drugs is like stumbling in the dark, hampering the transition to the clinic of these high-potential candidates. In a collaboration between MBB and the NMR spectroscopy group we study Lipid II binding antibiotics in membranes (or membrane models) by state-of-the-art solid state nuclear magnetic resonance spectroscopy, soon to be supported by the strongest commercial NMR magnet available: 1.2 GHz (expected to be operational in mid-2021).
This is a diverse project, including expression and purification of isotopically labeled peptides, enzymatic/chemical synthesis of Lipid II variants/fluorescent dye analogues, fluorescence spectroscopy, fluorescence microscopy, isothermal calorimetry (ITC), molecular modeling and of course solution/solid state NMR.
Schneider, T.; Kruse, T.; Wimmer, R.; Wiedemann, I.; Sass, V.; Pag, U.; Jansen, A.; Nielsen, A. K.; Mygind, P. H.; Raventos, D. S.; Neve, S.; Ravn, B.; Bonvin, A. M.; De Maria, L.; Andersen, A. S.; Gammelgaard, L. K.; Sahl, H. G.; Kristensen, H. H., Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Science 2010, 328 (5982), 1168-72.
Examples of our research:
Shukla, R.; Medeiros-Silva, J.; Parmar, A.; Vermeulen, B. J. A.; Das, S.; Paioni, A. L.; Jekhmane, S.; Lorent, J.; Bonvin, A. M. J. J.; Baldus, M.; Lelli, M.; Veldhuizen, E. J. A.; Breukink, E.; Singh, I.; Weingarth, M., Mode of action of teixobactins in cellular membranes. Nature Communications 2020, 11 (1), 2848.
Medeiros-Silva, J.; Jekhmane, S.; Paioni, A. L.; Gawarecka, K.; Baldus, M.; Swiezewska, E.; Breukink, E.; Weingarth, M., High-resolution NMR studies of antibiotics in cellular membranes. Nat Commun 2018, 9 (1), 3963.
Compared to Gram-positive bacteria, gram-negative bacteria are becoming more of a threat to man-kind in view of their increased resistance to antibiotics. One essential reason is that gram-negative bacteria possess an outer membrane. Lipopolysaccharide (LPS) is a main and essential component of this outer membrane. We are currently trying to specifically label the LPS of a gram-negative bacterium in order to follow its biosynthesis in real-time. When successful we will use this for screening fungal extracts that are active towards this biosynthesis pathway.
Peptidoglycan which is an important component of gram-negative bacteria cell wall can be labeled via by supplying certain precursors, which allows the specific labeling of the PG-layer in later stages of growth. . We will use this labeling in order to screen for compounds that are affecting the permeability barrier of the outermembrane, which would result in making gram-negative bacteria more sensitive to antibiotics.
The research in these programs involves both synthetic chemistry and biological chemistry. Some techniques that are used are: chemical synthesis, enzyme catalysis, column chromatography, NMR spectroscopy, Mass-spectroscopy, TLC, SDS-PAGE, plasmid isolation and transformation, fluorescence spectroscopy, fluorescence microscopy, solid phase synthesis ( using peptide synthesizer), LPS isolation and peptidoglycan isolation…
If you are interested in doing an internship with Yang, please feel free to contact him using the "Contact Yang Xu" button providing also a motivation. Students with a background in chemistry and/or biochemistry are welcome!