Correction to “Water-Binding Phospholipid Nanodomains and Phase-Separated Diacylglycerol Nanodomains Regulate Enzyme Reactions in Lipid Monolayers”

Correction to “Water-Binding Phospholipid Nanodomains and Phase-Separated Diacylglycerol Nanodomains Regulate Enzyme Reactions in Lipid Monolayers

Flow Cytometric Analysis To Evaluate Morphological Changes in Giant Liposomes As Observed in Electrofusion Experiments

Liposome fusion is a way of supplying additional components for in-liposome biochemical reactions. Electrofusion is a method that does not require the addition of fusogens, which often alter the liposome dispersion, and is therefore useful for repetitive liposome fusion. However, the details of electrofusion have not been elucidated because of the limitations surrounding observing liposomes using a microscope. Therefore, we introduced fluorescent markers and high-throughput flow cytometry to analyze the morphological changes that occur in liposome electrofusion. (i) The content mixing was evaluated by a calcein–Co²⁺–EDTA system, in which green fluorescence from dequenched free calcein is detected when the quenched calcein–Co²⁺ complex and EDTA are mixed together. (ii) Liposome destruction was evaluated from the decrease in the total membrane volume of giant liposomes. (iii) Liposome fission was evaluated from the increase in the number of giant liposomes. By applying the flow cytometric analysis, we investigated the effect of three parameters (DC pulse, AC field, and lipid composition) on liposome electrofusion. The larger numbers or higher voltages of DC pulses induced liposome fusion and destruction with higher probability. The longer application time of the AC field induced liposome fusion, fission, and destruction with higher probability. Higher content of negatively charged POPG (≥19%) strongly inhibited liposome electrofusion

Histograms of the M-m ratio for different concentrations of PEG lipid micelles.

<p>(a) 0 <i>μ</i>M, (b) 2.58 <i>μ</i>M, (c) 5.16 <i>μ</i>M, (d) 10.32 <i>μ</i>M, (e) 20.63 <i>μ</i>M, (f) 41.25 <i>μ</i>M, (g) 82.5 <i>μ</i>M, and (h) 165 <i>μ</i>M. Characteristic peaks were indicated as arrows.</p

Crystal Structure of a Membrane Stomatin-Specific Protease in Complex with a Substrate Peptide

Membrane-bound proteases are involved in various regulatory functions. A previous report indicated that the N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon <i>Pyrococcus horikoshii</i> is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis. Here, the crystal structure of 1510-N K138A in complex with a peptide substrate was determined at 2.25 Å resolution. In the structure, a 1510-N dimer binds to one peptide. The six central residues (VIVLML) of the peptide are hydrophobic and in a pseudopalindromic structure and therefore favorably fit into the hydrophobic active tunnel of the 1510-N dimer, although 1510-N degrades the substrate at only one point. A comparison with unliganded 1510-N K138A revealed that the binding of the substrate causes a large rotational and translational displacement between protomers and produces a tunnel suitable for binding the peptide. When the peptide binds, the flexible L2 loop of one protomer forms β-strands, whereas that of the other protomer remains in a loop form, indicating that one protomer binds to the peptide more tightly than the other protomer. The Ala138 residues of the two protomers are located very close together (the distance between the two Cβ atoms is 3.6 Å). Thus, in wild-type 1510-N, the close positioning of the catalytic Ser97 and Lys138 residues may be induced by electrostatic repulsion of the two Lys138 side chains of the protomers

Temporal changes of the mean and variance of the M-m ratio.

<p>Temporal changes of the mean and variance of the M-m ratio.</p

Histograms of the M-m ratio for different concentrations of PEG lipid micelles.

<p>(a) 0 <i>μ</i>M, (b) 2.58 <i>μ</i>M, (c) 5.16 <i>μ</i>M, (d) 10.32 <i>μ</i>M, (e) 20.63 <i>μ</i>M, (f) 41.25 <i>μ</i>M, (g) 82.5 <i>μ</i>M, and (h) 165 <i>μ</i>M. Characteristic peaks were indicated as arrows.</p

A schematic for vesicle image analysis.

<p>Vesicles are reacted with PEG lipid micelles to induce shape transformations and images are taken by a confocal microscope. Each vesicle image was binarized separately and approximated with an ellipsoid to measure the lengths of major and minor axes.</p

Reconstructed 3D images of a transforming lipid vesicle.

<p>(a) immediately after the addition of PEG lipid micelles, (b) after 2 min, (c) after 4 min (d) after 8 min. Numbers below images indicate the reduced volume <i>v</i>. The concentration of PEG lipid was 2.58 <i>μ</i>M.</p

A shape transformation of lipid vesicle induced by PEG lipid micelles.

<p>(a) A spherical vesicle transformed into (b) a disc-like oblate, (c) cylindrical prolate, and eventually divided into (d) two large and small vesicles.</p

<i>In Vitro</i> Membrane Protein Synthesis Inside Cell-Sized Vesicles Reveals the Dependence of Membrane Protein Integration on Vesicle Volume

Giant unilamellar vesicles (GUVs) are vesicles >1 μm in diameter that provide an environment in which the effect of a confined reaction volume on intravesicular reactions can be investigated. By synthesizing EmrE, a multidrug transporter from Escherichia coli, as a model membrane protein using a reconstituted <i>in vitro</i> transcription–translation system inside GUVs, we investigated the effect of a confined volume on the synthesis and membrane integration of EmrE. Flow cytometry was used to analyze multiple properties of the vesicles and to quantify EmrE synthesis inside GUVs composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. We found that EmrE was synthesized and integrated into the GUV membrane in its active form. We also found that the ratio of membrane-integrated EmrE to total synthesized EmrE increased with decreasing vesicle volume; this finding is explained by the effect of an increased surface-area-to-volume ratio in smaller vesicles. <i>In vitro</i> membrane synthesis inside GUVs is a useful approach to study quantitatively the properties of membrane proteins and their interaction with the membrane under cell-mimicking environments