Student projects - Antoinette Killian
SMA lipid membrane solubilization
Styrene-maleic acid (SMA) copolymers are able to solubilize lipid membranes into nanodiscs and thereby trap membrane proteins in their native lipid environment in a water-soluble form. With this method it is then possible to study not only properties of the protein itself, but also lipid-protein, protein-protein, and protein-ligand interactions. Membrane proteins make up a significant fraction of gene products and an even larger percentage of targets for pharmaceuticals. This highlights the importance of SMA as novel tool to study these highly sensitive and poorly soluble proteins.
Commercially available SMA polymers have the disadvantage that they have a very heterogeneous molecular weight distribution, as well as a non-uniform sequence distribution. Through collaboration with prof. Bert Klumperman from Stellenbosch University (South Africa, Department of Chemistry and Polymer Science) we have acquired for the first time well-defined and homogeneous polymers. Using these novel polymers it is being investigated what role polymer length and comonomer sequence have on the solubilization efficiency. The aim is to gain a deeper understanding of the mechanism of lipid membrane solubilisation by SMA to form nanodiscs. This will enable a more targeted approach when performing specific membrane protein solubilization studies for further downstream applications and it will help the development of new applications. To reach this goal the solubilization of lipid model membrane systems (liposomes) as well as biological membranes (E.coli) are being investigated. This is done by looking at various properties such as the kinetics, efficiency, and extent of solubilization. In addition, we aim to further characterize the resulting nanodiscs.
The SMALP field is rapidly growing and interest therein expanding.
The technique has the potential to revolutionize the study of membrane proteins and you may be part of this!
Depending on your individual interest and your precise project the techniques used may include:
- Electron Microscopy (EM)
- Dynamic Light Scattering (DLS)
- UV/Vis spectroscopy
- Infrared (IR) Spectroscopy
- Differential Scanning Calorimetry (DSC)
- Size-Exclusion Chromatography (SEC)
- Gas Chromatography (GC)
- Thin Layer Chromatography (TLC)
- Cell Culture
- Gel Electrophoresis (SDS-PAGE)
- Nuclear Magnetic Resonance (NMR)
- Organic Synthesis
For further reading see the following review:
Dörr JM, Scheidelaar S, Koorengevel MC, et al.
The styrene–maleic acid copolymer: a versatile tool in membrane research.
European Biophysics Journal. 2016;45:3-21. doi:10.1007/s00249-015-1093-y.
Islet Amyloid Peptide Project (IAPP)
Central in this line of research is the 37 amino acid long Islet amyloid peptide (IAPP) or Amylin. This peptide has our interest because it is involved in diabetes where it is responsible for pancreatic β-cell death. This peptide, as its name suggests, can form amyloid fibrils very similar to those seen in other amyloid diseases such as Alzheimer’s disease.
IAPP has been shown to interact with membranes and it seems very likely their cytotoxic effect is also dependent on this membrane interaction. Upon binding, conformational changes occur which have shown to be damaging.
Inhibitors that act on toxic IAPP species, or on the mature fibril are of interest because they could lead to the treatment of diabetes in the future. We are investigating several of these inhibitors and are trying to analyse how effective they are at stopping fibril growth and membrane damage.
We are using spectroscopic fluorescent techniques in the laboratory like Thioflavin T-assays to assess fibril growth, which is a standard technique in amyloid research. We are also specialized in looking at the influence of membranes on these processes and we use Calcein to see leakage of vesicle caused by IAPP induced damage.
The most recent published work from our group can be found here:
Saravanan, M. S., Ryazanov, S., Leonov, A., Nicolai, J., Praest, P., Giese, A., Winter, R., Khemtemourian, L. & Killian, J. A. (2019).
The small molecule inhibitor anle145c thermodynamically traps human islet amyloid peptide in the form of non-cytotoxic oligomers. Scientific Reports, 9(1), 19023. https://doi.org/10.1038/s41598-019-54919-z
If you are interested in doing a master internship with us please contact Barend Elenbaas. He is a PhD-candidate currently working on the IAPP-project.