Membrane-Fusogen Distance Is Critical for Efficient Coiled-Coil-Peptide-Mediated Liposome Fusion.

We have developed a model system for membrane fusion that utilizes lipidated derivatives of a heterodimeric coiled-coil pair dubbed E3 (EIAALEK)3 and K3 (KIAALKE)3. In this system, peptides are conjugated to a lipid anchor via a poly(ethylene glycol) (PEG) spacer, and this contribution studies the influence of the PEG spacer length, coupled with the type of lipid anchor, on liposome-liposome fusion. The effects of these modifications on peptide secondary structure, their interactions with liposomes, and their ability to mediate fusion were studied using a variety of different content mixing experiments and CD spectroscopy. Our results demonstrate the asymmetric role of the peptides in the fusion process because alterations to the PEG spacer length affect E3 and K3 differently. We conclude that negatively charged E3 acts as a "handle" for positively charged K3 and facilitates liposome docking, the first stage of the fusion process, through coiled-coil formation. The efficacy of this E3 handle is enhanced by longer spacer lengths. K3 directs the fusion process via peptide-membrane interactions, but the length of the PEG spacer plays two competing roles: a PEG4/PEG8 spacer length is optimal for membrane destabilization; however, a PEG12 spacer increases the fusion efficiency over time by improving the peptide accessibility for successive fusion events. Both the anchor type and spacer length affect the peptide structure; a cholesterol anchor appears to enhance K3-membrane interactions and thus mediates fusion more efficiently.

Interplay between Lipid Interaction and Homo-coiling of Membrane-Tethered Coiled-Coil Peptides.

The designed coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] are known to trigger efficient membrane fusion when they are tethered to lipid vesicles in the form of lipopeptides. Knowledge of their secondary structure is a key element in understanding their role in membrane fusion. Special conditions can be found at the interface of the membrane, where the peptides are confined in close proximity to other peptide molecules as well as to the lipid interface. Consequently, different structural states were proposed for the peptides when tethered to this interface. Due to the multitude of possible states, determining the structure solely on the basis of circular dichroism (CD) spectra at a single temperature can be misleading. In addition, it has not yet been possible to unambiguously distinguish between the membrane-bound and the coiled-coil states of these peptides by means of infrared (IR) spectroscopy due to their very similar amide I' bands. Here, the molecular basis of this similarity is investigated by means of site-specific (13)C-labeled FTIR spectroscopy. Structural similarities between the membrane-interacting helix of K and the homo-coiled-coil-forming helix of E are shown to cause the similar spectroscopic properties. Furthermore, the peptide structure is investigated using temperature-dependent CD and IR spectroscopy, and it is shown that the different states can be distinguished on the basis of their thermal behavior. It is shown that the two peptides behave fundamentaly differently when tethered to the lipid membrane, which implies that their role during membrane fusion is different and the mechanism of this process is asymmetric.

Biomaterials for mRNA delivery.

Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice.

Determination of oligomeric states of peptide complexes using thermal unfolding curves.

In their native form peptides are often found as oligomeric complexes, meaning they consist of more than one peptide chain. Coiled coils and helical bundles are common examples of such complexes. Their oligomeric state needs to be known precisely as this tremendously influences their biochemical and biophysical properties. The extensive analysis of circular dichroism spectroscopic data is commonly used to investigate the thermodynamics of binding and folding of these complexes. Here we present FitDis! an easy-to-use programme, which fits the most common two-state unfolding transition to the measured thermal unfolding curves of any oligomer of any stoichiometry. We demonstrate, with simulated and real examples, that the comparison of different stoichiometric models fitted to the same dataset reveals the oligomeric states of these complexes along with detailed thermodynamic information. This method will significantly ease the analysis of and increase the amount of information gained from, the thermal unfolding curves of peptide complexes.

Library of random copolypeptides by solid phase synthesis.

Random copolypeptides are promising and versatile bioinspired macromolecules of minimal complexity for studying their interactions with both living and synthetic matter. They provide the opportunity to investigate the role of, for example, total net charge and hydrophobicity through simply changing the monomer composition, without considering the effect of specific sequences or secondary structure. However, synthesizing large libraries of these polymers so far was prohibited by the time-consuming preparation methods available (ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides and enzymatic polymerization of amino acids). Here we report the automated solid phase synthesis (SPS) of a complete library of polypeptides containing Glu, Lys, and Ala monomers with excellent control over the degree of polymerization and composition and with polydispersity indices (PDIs) between 1.01 and 1.001, which is impossible to achieve by other methods. This method provides access to a library of polymers with a precisely defined total charge that can range from approximately -15 to +15 per chain and with a disordered conformation almost completely devoid of any secondary structure. In solution the polymers are largely present as unimers, with only the most hydrophobic polypeptides showing slight signs of aggregation. Our new approach provides convenient access to libraries of this versatile class of polymers with tunable composition, which can be used in a wide variety of physicochemical studies as a tool that allows systematic variation of charge and hydrophobicity, without the interference of secondary structure or aggregation on their performance.

Membrane interactions of fusogenic coiled-coil peptides: implications for lipopeptide mediated vesicle fusion.

Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of molecules. However, little is known about the actual fusion mechanism at the molecular level. In this study we examine a system that models the key features of this process. For the molecular recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called 'E' (EIAALEK)3 and 'K' (KIAALKE)3 were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides 'E', 'K', and their parallel coiled-coil complex 'E/K' with the phospholipid membranes and thereby mimicked the pre- and postfusion states, respectively. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramolecular chemistry and biomedicine.

Coiled-coil peptide motifs as thermoresponsive valves for mesoporous silica nanoparticles.

Coiled-coil peptide motifs were used as thermo-responsive valves for mesoporous silica nanoparticles (MSNs). The controlled release of a model drug as a function of temperature was demonstrated.

Controlling the rate of coiled coil driven membrane fusion.

Sets of complementary lipidated coiled-coil forming peptides that fuse membrane fusion have been designed. The influence of the coiled-coil motif on the rate of liposome fusion was studied, by varying the number of heptad repeats. We found that an increased coiled-coil stability of complementary peptides translates into increased rates of membrane fusion of liposomes.