Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement

Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural <i>Escherichia coli</i> phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon–Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3–5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of <i>E. coli</i> phospholipid extract used here

Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in <i>Escherichia coli</i>

Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in <i>Escherichia coli</i>, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from <i>Acholeplasma laidlawii</i> triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS <i>in vivo</i> together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed <i>in vitro</i> by ³¹P NMR binding studies using bicelles prepared with <i>E. coli</i> lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an <i>in vivo</i> signal for cells to create a “feed-forward” stimulation of lipid synthesis in <i>E. coli</i>. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis