Molecular insights into the mechanism of sensing and signal transduction of the thermosensor DesK

Abstract

The ability to sense and respond to environmental signals is essential for cell survival. Unraveling the molecular mechanisms underlying signaling processes remains a challenge, however. This thesis provides molecular insights into the mechanism of sensing and signal transduction of the thermosensor DesK. This thermosensor is embedded in the membrane and therefore, protein-lipid interactions are key to understanding its mechanism of sensing and signal transduction. In principle, these interactions can be investigated by studying the proteins in their native lipid environment. However, this is not straightforward and reducing the complexity allows more systematic studies. DesK was reduced in complexity by capturing both sensing and signaling properties in a single transmembrane helix: the minimal sensor DesK (MS-DesK). In addition, the native lipid membrane was replaced by a synthetic model membrane. Thus, to investigate what exactly is sensed by MS-DesK and to explore how the signal is transmitted, we could systematically vary the properties of the lipids, as well as properties of the transmembrane segment. This model system approach was exploited in chapters 2 and 3, where MS-DesK transmembrane segments were investigated with different membrane thickness, lipid phase and bilayer charge. The model systems were studied with circular dichroism, tryptophan fluorescence molecular modeling and complemented with in vivo functional studies. With the results of these complementary techniques we were able to construct a molecular model for sensing and signal transduction of DesK. To better mimic the in vivo situation, the synthetic lipids in the model membrane systems were replaced with lipid extracts from B. subtilis grown at relevant temperatures. These extracts were first characterized and we found temperature dependent regulation of the lipid acyl chain composition. Both the activity of a desaturase and an iso-anteiso switching mechanism were shown. In the model membrane systems with the B. subtilis lipids, no temperature dependent change in conformation of the MS-DesK transmembrane helix was observed. A possible explanation may be that the energetic barrier for reorientation of the helices is increased by the presence of branched side chains. In addition, owing to the attached cytosolic domain, the energetics of this barrier may be slightly different in vivo. The energetic barrier of the switch could for example be lowered because the linker region is more stable in vivo, reducing its interactions with the membrane. Finally, other proteins in the B. subtilis membrane affect the membrane properties, and thereby lower the energetic barrier for reorientation of the MS-DesK transmembrane segment. A very different model membrane system was used in chapter 5 to investigate the oligomerization state of MS-DesK. We explored the use of a styrene-maleic acid polymer (SMA), which solubilizes membranes into nanodiscs. Two approaches with these nanodiscs were investigated in this study. The first approach is based on the biochemical characterization of purified nanodiscs with transmembrane peptides. The second approach uses chemical cross-linking of the peptides in the nanodiscs. The results show that both of these approaches are promising tools for the characterization of oligomerization states. These methods, however, require further optimization.