Development of membrane-insertable lipid scrambling peptides: A time-resolved small-angle neutron scattering study

Abstract

Phospholipid transbilayer movement (flip-flop) in the plasma membrane is regulated by membrane proteins to maintain cell homeostasis and interact with other cells. The promotion of flip-flop by phospholipid scramblases causes the loss of membrane lipid asymmetry, which is involved in apoptosis, blood coagulation, and viral infection. Therefore, compounds that can artificially control flip-flop in the plasma membrane are of biological and medical interest. Here, we have developed lipid scrambling transmembrane peptides that can be inserted into the membrane. Time-resolved small-angle neutron scattering measurements revealed that the addition of peptides containing a glutamine residue at the center of the hydrophobic sequence to lipid vesicles induces the flip-flop of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine. Peptides without the glutamine residue had no effect on the flip-flop. Because the glutamine-containing peptides exhibited scramblase activity in monomeric form, the polar glutamine residue would be exposed to the hydrocarbon region of the membrane, perturbing the membrane and promoting the lipid flip-flop. These scrambling peptides would be valuable tools to regulate lipid flip-flop in the plasma membrane.

Small-Angle Neutron Scattering for Studying Lipid Bilayer Membranes

“…The peptide indolicidin speeds up lipid exchange between vesicles while also making them grow [ 250 ], as do the antimicrobial peptides Aurein 1.2, LL-37, and Lacticin Q [ 251 ] Other peptides have a similar effect [ 252 , 253 , 254 ]. A series of peptides designed to scramble lipids in a bilayer was studied by Nakao and coworkers using SANS and contrast-matching methods [ 255 ]. By tracking the integrated intensity in a specific q -range, they were able to measure the rate of change in the neutron scattering contrast and therefore the lipid exchange.…”

“… Peptide-enhanced exchange of lipids in vesicles [ 255 ]. ( A ) The integrated intensity as a function of time showing that two peptides engineered to scramble the lipids in the bilayer (4SQ and 4EQ) cause greater lipid exchange than when no peptide is present.…”

“…( B ) The decay in scattering contrast with time for the same system. The image is Figure 3 of reference [ 255 ] by Nakao et al, which was published under the Creative Commons Attribution License ( , accessed on 22 September 2022). The authors of reference [ 255 ] hold the copyright for the figure.…”

The Nanometer-Scale Proximity of Bilayers Facilitates Intermembrane Lipid Transfer

“…Polyethylene glycol (PEG) is known to induce the fusion of lipid vesicles and cells. − The presence of PEG induces depletion attraction between large obstacles owing to the excluded volume effect, resulting in their aggregation. , As PEG indirectly causes attraction between vesicles, this technique can be used to analyze the properties of bilayers adjacent to each other without introducing docking reagents into the membrane. Here, we investigate lipid transfer between vesicles brought into proximity by PEG using time-resolved small-angle neutron scattering (TR-SANS), a technique first established by our group for evaluating interbilayer and transbilayer transfer (flip-flop) of lipids. − The thermodynamic activation parameters of lipid transfer were derived from the temperature dependence of the transfer. Notably, the inclusion of PEG-conjugated lipids into the vesicles manipulated the interbilayer distance of the aggregated vesicles, which was quantitatively analyzed using SANS.…”

Energetic and Structural Insights into Phospholipid Transfer from Membranes with Different Curvatures by Time-Resolved Neutron Scattering

“…We have demonstrated that time-resolved small-angle neutron scattering is an effective method for evaluating the intervesicular transfer and flip-flop of phospholipids. − This technique has been used to measure not only the spontaneous transfer of phospholipids − but also lipid transfer via phospholipid transfer proteins and flip-flop via synthetic or naturally occurring peptides. − Since the transfer of phospholipids between membranes occurs by dissociation from the outer leaflet of the membrane, the energetic information on the outer leaflet of the bilayer can be extracted. In this study, this technique was applied to measure the lipid dissociation kinetics of vesicles of different sizes to evaluate the effect of membrane curvature on phospholipid stability in lipid bilayers.…”