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Figure 1 shows the process of membrane fusion as mediated by the SNARE protein complex.

SNARE-Mediated Membrane Fusion and dPEG®, Part 3

Part 3: Modeling Membrane Fusion with SNARE Protein Analogs

Click the links for Part 1 and Part 2 of this three-part series.

Membrane fusion is a universal process in living organisms and is critical to proper cellular function. Examples of membrane fusion events include exocytosis, fertilization, envelopment of infecting viruses, and transport of proteins through the Golgi stack. Though essential, membrane fusion is not spontaneous because free energy is required to overcome steric hindrances, electrostatic repulsions, and hydration shells between two membranes as they move toward each other prior to fusion.

In cells membrane fusion is facilitated by a protein superfamily known as SNARE (for Soluble N-ethylmaleimide-sensitive-factor Attachment Receptor), of which there are 36 members in humans. The best studied SNARE-mediated membrane fusion process is exocytosis in neural cells. Indeed, the first proteins of the human SNARE family to be identified were synaptobrevin, syntaxin, and synaptosome-associated protein 25 (SNAP-25). Figure 1 shows a SNARE-mediated exocytosis.

 

Figure 1 shows the process of membrane fusion as mediated by the SNARE protein complex.
Figure 1: Membrane Fusion via SNARE Protein Complex Mediation, By Danko Dimchev Georgiev, M.D. [GFDL or CC-BY-SA-3.0], via Wikimedia Commons
 

Because the native SNARE-mediated process is so complex, numerous research groups have worked to create simplified artificial systems that mimic SNARE-mediated membrane fusion. Numerous systems have been developed using both targeted and non-targeted membrane fusion. Pawan Kumar, Samit Guha, and Ulf Diederichsen have written an excellent review of these SNARE mimetic systems, focusing on targeted membrane fusion. The review is listed in the References section below with a link to the paper, which is behind a paywall.

Most of the targeted SNARE mimetic systems discussed in the review consist of a lipophilic anchor (cholesterol, POPE, and DOPE are discussed), a flexible linker, and a recognition sequence. The flexible linker most often used is a PEG linker, typically a PEG12, though PEG3 has also been used. The purpose of the PEG linker is to site the recognition sequence away from the lipid membrane but to maintain flexibility so that the recognition sequence can move freely in space until it pairs with its target sequence. Example systems using Quanta BioDesign's dPEG®12 linkers (most notably PN10283, Fmoc-N-amido-dPEG®12-acid) have been discussed in Part 1 and Part 2 of this series.

 

 How dPEG® Can Help You

Quanta BioDesign's dPEG® linkers provide the traditional advantages of PEG (water solubility, low immunogenicity, reduced aggregation and precipitation of proteins) but with a critical difference. Traditional PEGs are dispersed, but Quanta BioDesign's patented dPEG® constructs are discrete molecules. Constructs made from dispersed PEGs confound analysis because of the intrinsic heterogeneity of the PEG. Each of Quanta BioDesign's dPEG® constructs is a single molecular entity. There is no range of linker sizes or molecular weights. Thus, products made from dPEG® are well-defined, easily characterized, and amenable to standard analytical techniques.

Whether you are studying SNARE protein-mediated membrane fusion, building a new drug delivery platform, or creating a new peptide or protein drug, if you need PEG, then you need dPEG®. Quanta BioDesign invented and patented the dPEG® construct, and we manufacture our products in the United States.

 

References and Additional Reading

(1) Pawan Kumar, Samit Guha, and Ulf Diederichsen. SNARE protein analog-mediated membrane fusion. J Peptide Sci (2015), 21(8), 621-629. DOI: 10.1002/psc.2773

(2) Lando L. G. Schwenen, et al. Resolving single membrane fusion events on planar pore-spanning membranes. Scientific Reports (2015), 5:12006. DOI: 10.1038/srep12006

(3) Jörg Malsam and Thomas H. Söllner. Organization of SNAREs within the Golgi Stack. Cold Spring Harb Perspect Biol (2011), 3:a005249. DOI: 10.1101/cshperspect.a005249

(4) Yu A. Chen and Richard H. Scheller. SNARE-mediated membrane fusion. Nature Reviews Mol. Cell Biol (February 2001), 2, 98-106.

(5) Reinhard Jahn, Thorsten Lang, and Thomas C. Südhof. Membrane Fusion. Cell (February 21, 2003), 112, 519-533. DOI:10.1016/S0092-8674(03)00112-0

(6) Joseph G. Duman and John G. Forte. What is the role of SNARE proteins in membrane fusion? Am J Physiol Cell Physiol 285: C237–C249, 2003; DOI: 10.1152/ajpcell.00091.2003.

(7) Reinhard Jahn and Richard H. Scheller. SNARES — Engines for Membrane Fusion. Nature Reviews Mol Cell Biol (September 2006), 7, 631-643. DOI:10.1038/nrm2002

 

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.

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Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

SNARE-Mediated Membrane Fusion and dPEG®, Part 2

Part 2: Controlling Liposome Fusion Using SNARE Protein Mimics

For Part 1 of this three-part series, click here. For part 3, click here.

Introduction

Continuing earlier work, researchers at the laboratory of Alexander Kros examined the parameters that affect membrane fusion in liposomes composed of the minimal model developed in their earlier paper (1). In this paper (2), the research group demonstrated that, using the model system previously developed, "the rate and extent of fusion and the number of fusion rounds can be manipulated by adjusting the fusogen and liposome concentrations" (quote from the abstract).

The SNARE protein model for membrane fusion developed by the research group contained 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) connected to one of two oligopeptides (LPE or LPK) via a dPEG®12 linker derived from PN10283, Fmoc-N-amido-dPEG®12-acid, where the PEG linker served as a means to provide a flexible spacer between the DOPE and the oligopeptides. See Figure 1, below. The coiled-coil forming oligopeptides each contain three-heptad repeats, and when populations of liposomes containing the LPE and LPK constructs are mixed, they associate and lead cleanly to liposome fusion of the two different liposome populations. As discussed in the earlier post on this subject, the model system developed by Kros and colleagues is an accurate, minimalist model of SNARE-mediated liposome fusion.

 

Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.
Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

For this research, the authors characterized the influence on liposome fusion of lipopeptide concentration, lipid concentration, and lipids of positive curvature. The authors also described how liposome fusion in the model system varies with liposome collision, docking, and lipid mixing rates.

 

 

How Liposome Fusion Works

The group first studied whether liposome fusion was targeted (that is, molecular recognition between two different fusogens) was required for fusion to occur or non-targeted (no molecular recognition required). This analysis was carried out by mixing populations of liposomes not modified with either LPE or LPK with populations of liposomes carrying either LPE or LPK. In these experiments, no liposome fusion occurred. However, in experiments in which LPE-modified liposomes were mixed with LPK-modified liposomes, liposome fusion occurred. This indicated that molecular recognition between the LPK and LPE peptides was required for liposome fusion to occur; hence, the fusion was targeted.

Next, the researchers examined the effect of concentration of the two fusogens on liposome fusion. By mixing decreasing proportions (mole%) of the lipopeptide fusogens in a fixed amount of total lipids, the scientists were able to determine the lower limit and optimal amount of fusogens required for liposome fusion. By this method, it was determined that 0.05 mole% lipopeptide was the lower limit of fusogens required for at least one round of liposome fusion to occur. The optimal amount of fusion was found to be 0.75 mole% LPE or LPK incorporated into the liposome membranes. Circular dichroism data demonstrated that the lipidated oligopeptides (LPE and LPK) were most likely distributed evenly across the membrane surface at 0.75 mole%.

 

The general trend of increasing membrane fusion rates with increasing proportions of fusogens arises because two liposomes that diffuse into close proximity are more likely to be displaying complementary peptides in the correct orientation for binding in the approaching area, and hence are more likely to dock and undergo fusion. (2) at page 1048.

 

Above 0.5 mole% lipopeptide concentration, the researchers found that completely lipid mixing occurred rapidly. Large particles ("giant liposomes") and clusters of docked and fused particles formed, and the particles sedimented in a matter of hours. Conversely, at 0.25 mole% or less lipopeptide concentration, the particles that form from liposome fusion are initially small, increase in hydrodynamic diameter size, then decrease again over time before plateauing "at the size corresponding to the fusion of two liposomes in which the volume is conserved rather than the outer surface area" (2, p. 1050). This indicates that the SNARE mimetic liposome fusion is "clean," meaning that no loss of content occurs during fusion.

The research group analyzed whether lipid concentration affected membrane fusion, and if so, by how much. Using FRET experiments, the group found that complete lipid mixing was more efficient if there were more fusogens (the DOPE-PEG12-oligopeptides) on fewer liposomes than if there were fewer fusogens on more liposomes. A lower limit of 0.025 mM lipids (0.25 µM lipopeptide) was established as the lower limit of lipid concentration at which liposome fusion occurred efficiently.

Finally, the group examined the effect of changing lipid curvature on liposome fusion. Using the positive curvature lipid lysophosphatidylcholine (LPC) instead of DOPE altered the shape of the liposomal surface and prevented formation of the so-called "stalk intermediate" that is required in SNARE-mediated fusion (the stalk intermediate requires a negative curvature in order to form). With as little as 15 mole% LPC incorporated into liposomes, the researchers found that lipid mixing decreased dramatically; however, docking (the first step in liposome fusion) was not inhibited, but hemifusion and full fusion were prevented from occurring.

The research performed provided detailed understanding of how the reduced SNARE model works to promote liposome fusion. This detailed degree of understanding is important for developing applications of liposome fusion (or membrane fusion) such as targeted delivery of drugs to cells and controlled mixing of reagents at nanoliter volumes, such as would occur in a lab-on-a-chip application.

 

dPEG® Role in This Research

As mentioned in an earlier post, the DOPE-PEG12-LPE/LPK constructs used PN10283, Fmoc-N-amido-dPEG®12-acid to create the flexible extension between the lipid membrane surface and the oligopeptides. 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. Second, PEG reduces aggregation and precipitation of biomolecules. Third, PEG diminishes the immunogenicity of molecules to which it is conjugated. Conventional PEG, though, is polydispersed, meanign that it 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.

We encourage you to look at all our products and find what you need for your next research project. 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) Hana Robson Marsden, Alexander V. Korobko, Tingting Zhen, Jens Voskuhl, and Alexander Kros. Controlled liposome fusion mediated by SNARE protein mimics. Biomaterials Science (2013), 1, 1046-1054. DOI: 10.1039/c3bm60040h

 

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.

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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 products 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.

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