| Membrane proteins are abundant in nature and play a key role in many essential life processes. They typically span the membrane with one or more hydrophobic segments. Temporal changes in properties of such transmembrane (TM) segments often are a prerequisite for functional activity of membrane proteins. For example, the propagation of a signal through TM segments is one of the initial steps within many complex signaling cascades. However, very little is known about the molecular nature of this important step in signaling. Recent discoveries on a regulation system of membrane fluidity by the molecular thermosensor DesK in Bacillus subtilis now open up the way to elucidate for the first time in molecular detail how the TM segments of a signaling protein act as sensor and how they are able to transmit this information to the transcriptional system. The regulation system was discovered by the group of Diego de Mendoza [1]. The system consists of a membrane-embedded sensor, DesK, which reacts to temperature changes by regulating the expression of a desaturase Des, that inserts into the membrane and introduces unsaturated bonds in the lipids, thereby regulating membrane fluidity. The recent discoveries in this group that now allow elucidation of the pathway are (i) the fascinating finding that both sensing and transmission of DesK, which has five TM segments, can be captured into one single TM segment (the so-called ?minimal sensor?) and (ii) that DesK retains its functionality even when reconstituted in pure lipid vesicles and hence that no other protein components are involved in either sensing or signaling. The DesK system thus allows minimization of a complex biological phenomenon to a perfectly simple functional system of the behavior of a single TM segment in a lipid bilayer. In principle, there are many ways in which TM segments may sense changes in temperature and propagate this into a signal for transcription. For example, as a response to changes in lipid fluidity they may undergo changes in tilt or rotation angle, in conformation, or in precise positioning at the lipid/water interface or they may oligomerize. Such changes in the TM segments could bring about conformational changes at the cytoplasmic side of the membrane and/or result in changes in accessibility of specific protein sites to certain enzymes, leading to a response within the cell. The availability of a simplified functional signaling system now allows us to elucidate the molecular nature of the changes in properties of the TM segments underlying both the sensing and the transmission mechanism by using well-defined model membranes of synthetic lipids and designed peptides corresponding to TM segments of functional and non-functional minimal DesK sensors. Within this project, the structural and dynamic properties of these peptides will be analyzed as function of temperature upon reconstitution in synthetic lipid bilayers. A range of biophysical techniques, including computational approaches, will be used to study how bilayer thickness and lipid fluidity influence peptide conformation, tilt angle, oligomerization, and positioning of the peptide at the interface. The results will tell us which properties of the peptides and of the lipids are likely to be responsible for sensing and for signaling. In addition, lipid extracts will be prepared of B. subtilis. Analysis of the behavior of the peptide in these extracts, combined with determination of the properties of these lipids (e.g. phase behavior, fluidity, thickness) will allow us to further link the sensing and transmission signals observed in model membranes with the situation in vivo. Finally, we will collaborate intensively with the group of de Mendoza, who is developing a library of functional and non-functional DesK minimal sensors which will guide the design of all synthetic peptides. Vice versa, any hypothesis generated from experiments on the synthetic peptides will be tested on mutant DesK minimal sensors in the group of de Mendoza. Together these approaches can be expected to result in a robust and detailed model for the molecular mechanism of sensing and transmission of the DesK minimal sensor. The proposed research directly builds on our expertise, built over the years, on the interactions between synthetic, designed transmembrane peptides and lipids in model membrane systems [e.g. 2,3 and references therein] and on our expertise in analyzing lipid composition and lipid phase behavior [e.g. 4,5]. The DesK system provides a unique opportunity to apply and extend this knowledge to study a highly relevant, but complex biological phenomenon: sensing and regulation of membrane fluidity by a bacterial thermosensor. |
