Chemical structure of PN10283, Fmoc-N-amido-dPEG®12-acid

SNARE-Mediated Membrane Fusion and dPEG®, Part 1

Part 1: A Reduced SNARE Model for Membrane Fusion

The cells of all living things depend on membrane fusion for intra- and intercellular transport of molecules. In both cellular membrane fusion and intracellular vesicle fusion, the fusion process is controlled and guided by SNARE proteins. SNARE is an acronym for Soluble NSF Attachment Protein Receptor. NSF stands for N-ethylmaleimide-Sensitive Factor. Reviews of SNARE protein structure and function can be found here, here, here, and here. An example of a SNARE protein is synaptobrevin. Click part 2 and part 3 to read the other pieces in this series. Figure 1 shows a diagram of the process of membrane fusion. Figure 2 shows detail of two membranes fusing with SNARE proteins involved in the process. Both images are from Wikipedia and have a creative commons license.

FIgure 1 shows the process of lipid bilayer fusion such as is found in cell membrane fusion and vesicle fusion.
Figure 1: Lipid Bilayer Fusion Process (image from Wikipedia; image is not altered from the original)

Lipid bilayer fusion by MDougM. Licensed under Public Domain via Creative Commons

Figure 2 shows the exocytosis machinery in membrane fusion.
Figure 2: The Exocytosis Machinery Showing How Membrane Fusion Works (image from Wikipedia.com via a Creative Commons License; image is not altered from the original).

Exocytosis-machinery by Danko Dimchev Georgiev, M.D. – http://en.wikipedia.org/wiki/Image:Exocytosis-machinery.jpg. Licensed under CC BY-SA 3.0 via Creative Commons

Membrane Fusion Modeling

It is critical to model membrane fusion in order to understand and control it. A good model of membrane fusion via SNARE proteins helps scientists understand the fusion process and may permit them to conduct membrane fusion under highly controlled conditions to carry out advanced applications in medical diagnostics and therapeutics. To create a good model for membrane fusion, researchers must find the minimal biochemical machinery needed to reproduce membrane fusion fully in a test system.

In 2009, researchers at the lab of Alexander Kros, Leiden Institute of Chemistry, Leiden University, The Netherlands, took a significant step toward that goal(1). In the process, they used Quanta BioDesign’s product PN10283, Fmoc-N-amido-dPEG®12-acid as part of the model system(2). Starting with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), Kros’ group coupled PN10283 to DOPE. The DOPE-PEG construct was then coupled to one of two lipidated oligopeptides, designated LPE and LPK. Each peptide contains a three-heptad repeat (designated E and K) that are the shortest known coiled-coil pair to form a stable heterodimer. See Figure 3, below.

Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).
Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).

The purpose of this design was to mimic the essential functions of a SNARE protein complex but to minimize the number of different elements needed to direct membrane fusion. The DOPE portion of the construct thus spontaneously anchors the LPE and LPK oligopeptides into the membrane. The dPEG® linker provides a short, flexible spacer that extends the oligopeptides away from the membrane. The two peptides themselves provided the driving force to bring the two membranes together, allowing for membrane fusion.

Using circular dichroism and fluorescence resonance energy transfer (also called Förster resonance energy transfer, known popularly by the acronym FRET) assays with liposome constructs, the researchers were able to distinguish and analyze three distinct stages in membrane fusion. Each stage required an energy input in order to proceed. In the first stage, the liposomes were brought into close proximity. A FRET assay using nitrobenzofuran and lissamine rhodamine dyes attached to the lipid bilayer surfaces showed that the LPE/LPK coiled coil complex that forms spontaneously when the liposomes are in close proximity provides sufficient energy to bring the liposomes together and initiate lipid mixing of the outer lipid layers (known as hemifusion). Further FRET experiments showed that the fusion process initiated by the LPE/LPK coiled coil complex provided enough energy to drive the fusion process to the point of mixing both the outer and inner lipid layers. Furthermore, once the lipid layers were mixed, the contents of the liposomes were observed to mix also.

Conclusions

The approach taken in this paper allowed the research group to build a minimal model of membrane fusion. Indeed, the authors concluded,

“The reduced SNARE model presented herein has been shown to meet all of the characteristics of native membrane fusion, and this similarity combined with the ease of use makes the system a true minimal model for SNARE mediated membrane fusion.” (1) at page 2333

Quanta BioDesign Helps You Build Complex Constructs

This research shows the power of modeling even for complex systems such as membrane fusion. Moreover, this research demonstrates the flexible utility of dPEG® products, in this case PN10283, Fmoc-N-amido-dPEG®12-acid.

If you need to add PEG to your supramolecular construct, why not consider dPEG®? There are numerous advantages to adding PEG to your peptide or protein construct. First, PEG is amphiphilic, providing enhanced solubility in both water and organic solvents. Second, PEG has been proven to reduce aggregation and precipitation of biomolecules. Third, PEG reduces immunogenicity of molecules to which it is conjugated.

Dispersity is the key advantage of using Quanta BioDesign’s dPEG® products instead conventional PEG products. Conventional PEG is dispersed. Quanta BioDesign’s dPEG® products are single molecular weight compounds. That is, rather than having a single discrete molecular size and weight, a conventional PEG is composed of numerous PEG molecules. The “weight” of a conventional PEG is expressed as an average molecular weight across the range of sizes. Quanta BioDesign’s dPEG® constructs — designed, patented, and manufactured in the United States — are single molecular weight PEG molecules functionalized with various chemical moieties. Whereas conventional PEGs confound analysis by presenting an intractable mixture of different size molecules, dPEG® constructs enhance analysis by being a single, discrete molecule that can be accurately characterized using ordinary analytical chemistry tools.

Check out our latest catalog in PDF format or browse online. You’ll be pleasantly surprised at what you can do with dPEG®!

References

(1) Hana Robson Marsden, Nina A. Elbers, Paul H. H. Bomans, Nico A. J. M. Sommerdijk, and Alexander Kros. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed (2009), 48, 2330-2333. DOI: 10.1002/anie.200804493

(2) The dPEG® was purchased from Iris Biotech GmbH, one of Quanta BioDesign’s highly valued distributors. Iris Biotech is located in Marktredwitz, Germany.

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign, and on Google+ at https://plus.google.com/+RobertWoodman.