Most biopolymers like proteins, DNA, and RNA molecules function inside the cell. We are interested in studying biomolecular structure, function and aggregation directly in cellular environments. Therefore, special in-cell techniques are used and the results are interpreted by comparative in vitro experiments in cell-like environments and dilute solutions (Fig. 1).
The Ebbinghaus group is interested in studying how cells keep their biomolecules folded in space (i.e. spatially inside mammalian cells) and time (i.e. age-dependent changes). Therefore, the group studies how physicochemical factors (such as crowding, confinement, hydration) or biological factors (such as chaperones, cell stresses) modulate biomolecular fold and function.
One factor that alters folding in cells is macromolecular crowding. Although it was assumed that crowding effects are mainly mediated by entropic excluded volume effects, the group could show that enthalpic stabilization and entropic destabilization effects determine the protein folding stability (Senske et al., JACS, 2014). Including osmolytes and electrolytes, the lab developed a novel model to describe cosolute effects based on their thermodynamic fingerprints. Therefore, the ‘stability curve’ of proteins is analyzed over a wide range of temperatures rather than focusing on a single point of the stability curve (e.g. the melting point Tm). Regarding the multiple intersections of the respective stability curves for different salts, a temperature dependent model of Hofmeister effects was developed that replaced the common view of a simple homologous series (Senske et al., PCCP, 2016).
The group further developed biosensors to quantify crowding effects directly in living cells (Gnutt et al.,Angew. Chem. Int. Ed., 2015, Gao et al., Angew. Chem. Int. Ed., 2016). These sensors are able to report on the different contributions of crowding effects such as excluded volume, non-specific interaction or water activity. Their net balance can be perturbed for example by osmotic stress. Further, the cellular environment imposes stabilizing or destabilizing effects to nucleic acids while the folding stability is dependent on subcellular localization (Gao et al., Angew. Chem. Int. Ed., 2016). Such variations could be utilized by the cells to locally adjust the energy landscape for RNA folding.
Similar effects can be observed for proteins, where most destabilizing conditions lead to protein aggregation. The lab developed a new technology to measure aggregation kinetics of Huntingtin exon-1 (Vöpel et al., JACS, 2017, Büning et al., PCCP, 2017) or human islet polypeptide hIAPP (Gao et al.,PCCP, 2015) directly on the cellular level by using fast laser-induced temperature jumps in combination with microscopy (Vöpel et al., JACS, 2017, Vöpel et al., Chem. Commun., 2015). This allows analyzing cellular factors that either promote (e.g. osmotic stress) or prevent (e.g. chaperone interactions or protective osmolytes) protein aggregation. Using the in-cell aggregation assay, the group explores new strategies to interfere with protein aggregation developing new therapeutic intervention against neurodegenerative diseases (Vöpel et al., JACS, 2017).
Please note: Once you watch the video, data will be transmitted to Youtube/Google. For more information, see Google Privacy.
