Modulation of Concentration Fluctuations in Phase-Separated Lipid Membranes by Polypeptide Insertion

Abstract

The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures corresponding to the gel-fluid phase coexistence region the concentration fluctuations drastically decrease, and we observe domains in the giant unilamellar vesicles, which mainly disappear by the incorporation of 2 to 5 mol% GD. Further, the lipid matrix has the ability to modulate the conformation of the inserted polypeptide. The balance between double-helical and helical dimer structures of GD depends on the phospholipid chain length and phase state. A large hydrophobic mismatch, such as in gel phase one-component DSPC bilayers, leads to an increase in population of double-helical structures. Using an effective molecular sorting mechanism, a large hydrophobic mismatch can be avoided in the DMPC-DSPC lipid mixture, which leads to significant changes in the heterogeneous lipid structure and in polypeptide conformation.

Tuning Membrane Thickness Fluctuations in Model Lipid Bilayers

“…Indeed, in this region we expect the two lipids to be mostly segregated into DSPC gel domains and DMPC fluid domains (40,41). From our previous results, we expect only the fluid DMPC domain fluctuations to be measurable and their amplitude to be z0.4 nm, which matches the amplitude measured in this coexistence region within the experimental error.…”

A Novel Heavy‐Atom Label for Side‐Specific Peptide Iodination: Synthesis, Membrane Incorporation and X‐ray Reflectivity

“…A b 5.6 -helix consisting of twelve amino acids with a pitch of 5.1 would exhibit a length of approximately 29 while the corresponding b 6.3 -helix with a 3.3 pitch would feature a length of approximately 21 . [46][47][48][49][50] Considering these facts in addition to the CD results and previous studies, the distance indicated in the electron density difference curves by the major peaks that are located right beneath the lipid head-groups additionally suggest the existence of a membrane-spanning b 5.6 -helix. Especially because of the recently reported crystal structure where the peptides of the present motif seem to be slightly shorter than the typical b 5.6 -helical structure, maybe due to the steric effects of the side chains and by means of hydrophobic mismatch an extension of the considered species of 0.8 is highly reasonable.…”

Partitioning of Dual-Lipidated Peptides into Membrane Microdomains: Lipid Sorting vs Peptide Aggregation

“…The circular shape of the domains observed here ( Figure 5B), which contrasts with the gel-fluid type domains seen in Figure 2, is typical for minimizing the area-to-perimeter ratio for isotropic phases in equilibrium. This kind of shape is also observed in model raft mixtures 42 that are thought to consist of liquid-ordered/liquid-disordered domains. These findings can be explained by a lipid sorting mechanism and phase separation on the vesicle surface induced by the polypeptide.…”

“…11 Recently, we studied giant unilamellar vesicles (GUVs) of the pure lipid mixture using two different fluorophores (Laurdan and N-Rh-DPPE) in two-photon excitation fluorescence microscopy studies. 42 In the case of N-Rh-DPPE, the different probe partitioning between gel and fluid domains discriminates between fluid and gel-state domains. In the fluid phase of the lipid bilayer system, at T g 49°C, the images obtained with N-Rh-DPPE as fluorophor show that the fluorescent molecules are distributed homogeneously on the vesicle surface ( Figure 2).…”