Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the ...lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EG
n
) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (L
o
) and liquid disordered (L
d
) phase coexisting membranes. Partitioning coefficients (
K
p L
o
/L
d
) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EG
n
-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving
K
p
of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.
Short PEG spacers effectively decouple headgroup and receptor-ligand interactions from the membrane allowing packing order of the lipid tails to direct partitioning of lipids to specific membrane phases.
Cellular membranes are densely covered by proteins. Steric pressure generated by protein collisions plays a significant role in shaping and curving biological membranes. However, no method currently ...exists for measuring steric pressure at membrane surfaces. Here, we developed a sensor based on Förster resonance energy transfer (FRET), which uses the principles of polymer physics to precisely detect changes in steric pressure. The sensor consists of a polyethylene glycol chain tethered to the membrane surface. The polymer has a donor fluorophore at its free end, such that FRET with acceptor fluorophores in the membrane provides a real-time readout of polymer extension. As a demonstration of the sensor, we measured the steric pressure generated by a model protein involved in membrane bending, the N-terminal homology domain (ENTH) of Epsin1. As the membrane becomes crowded by ENTH proteins, the polymer chain extends, increasing the fluorescence lifetime of the donor. Drawing on polymer theory, we use this change in lifetime to calculate steric pressure as a function of membrane coverage by ENTH, validating theoretical equations of state. Further, we find that ENTH’s ability to break up larger vesicles into smaller ones correlates with steric pressure rather than the chemistry used to attach ENTH to the membrane surface. This result addresses a long-standing question about the molecular mechanisms of membrane remodeling. More broadly, this sensor makes it possible to measure steric pressure in situ during diverse biochemical events that occur on membrane surfaces, such as membrane remodeling, ligand–receptor binding, assembly of protein complexes, and changes in membrane organization.
Random orientation of molecules within a sample leads to blurred observations of chemical reactions studied from the laboratory perspective. Methods developed for the dynamic imaging of molecular ...structures and processes struggle with this, as measurements are optimally made in the molecular frame. We used laser alignment to transiently fix carbon disulfide molecules in space long enough to elucidate, in the molecular reference frame, details of ultrafast electronic-vibrational dynamics during a photochemical reaction. These three-dimensional photoelectron imaging results, combined with ongoing efforts in molecular alignment and orientation, presage a wide range of insights obtainable from time-resolved studies in the molecular frame.
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