In vitro and in vivo supramolecular modification of biomembranes using a lipidated coiled-coil motif.

The molecular building blocks available in biological systems self-assemble into defined structures in an extremely controlled manner. These structures must be flexible and adaptive to the environment in order to carry out their function in a regulated manner. Therefore, nature uses multiple weak interactions (e.g. hydrogen bonding and van der Waals interactions) to act as the glue to hold these structures together. When many weak interactions cooperatively combine, relatively stable entities are produced, which retain the ability to respond to external stimuli such as fluctuations in ion concentration, pH, and temperature. For many years, nature has been a source of inspiration for supramolecular chemistry. Scientists typically follow the bottom-up approach and design relatively simple molecules which assemble into functional materials with well-defined properties. Recent progress has resulted in molecular systems that are responsive to multiple stimuli and are therefore highly controlled, emulating nature ever more closely. A relatively new development is the application of supramolecular constructs in in vitro and in vivo environments to directly study and influence biological processes in live cells. Chemically tailored systems can be integrated into cell membranes, for example. This enables the modification or regulation of cellular behavior through external artificial signals. There are two approaches for introducing chemical species into a cell membrane by supramolecular chemistry: 1) specific binding of guest molecules to membrane-anchored biomolecules such as native proteins and 2) nonspecific labeling of membranes with the aid of hydrophobic and electrostatic interactions or through a chemical crosslinker. Lipidated peptides are particularly good candidates for application in biological systems as their aggregation behavior can be controlled by carefully balancing the hydrophobicity of the anchor and the hydrophilicity of the cargo; this aids the incorporation of lipidated peptides into membranes. Here we describe the use of a coiled-coil motif as the peptide segment, a highly specific recognition system that can be introduced into live cells. The coiled-coil motif acts as molecular Velcro and can thus be used to link distinct molecular constructs. An example of the specific labeling of proteins through coiled-coil formation was recently supplied by Matsuzaki et al. Surface modification through the nonspecific binding of polymers to cell membranes has also been studied, for example by Ijiro et al. Lipid-grafted polymers adhere to cell membranes and could potentially act as a scaffold to which a wide range of functional moieties could be attached, thereby intervening in the chemistry of the cell. Furthermore, cationic graft copolymers have also been shown to interact electrostatically with cell membranes, resulting in chemically altered cell membranes. Although these examples illustrate that in vitro membrane functionalization is a highly rewarding strategy, there are currently no examples of efficient in vivo strategies. Therefore, it is our goal to transiently modify lipid membranes through specific supramolecular interactions in in vitro and in vivo environments. For this purpose, we use a pair of complementary coiled-coil-forming lipidated peptides (E and K peptides) to specifically introduce a noncovalent and bio-orthogonal recognition motif to biological membranes (Scheme 1). Here, we describe a generic supramolecular tool which allows us to rapidly and efficiently form coiled-coil motifs at the surface of biological membranes. This is of interest as a wide range of molecular constructs can be introduced to the surface of the cell in this way. Coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] [12] were first covalently conjugated to PEG12 spacers (PEG= polyethylene glycol). Subsequently, a cholesterol moiety was coupled to the pegylated peptides yielding CPE and CPK (Scheme 1A). The cholesterol moiety allows for the immediate insertion of the lipidated peptides into membranes through hydrophobic interactions and the PEG12 moiety was incorporated to aid efficient molecular recognition between the peptide segments E and K. Recently we showed that upon the addition of micellar solutions of either CPE or CPK to plain liposomes, the lipidated peptides spontaneously inserted into liposomal membranes. In the current study, CHO cell membranes (CHO=Chinese hamster ovary) and the skin of zebrafish embryos were modified with coiled-coil-forming peptides by the addition of a micellar solution of CPE or CPK, resulting in immediate incorporation of these amphiphiles into the membranes. Subsequently, the complementary peptide was added, result[*] M. Sc. H. R. Zope, M. Sc. F. Versluis, Dr. J. Voskuhl, Dr. A. Kros Soft Matter Chemistry, Leiden Institute of Chemistry Leiden University P.O. Box 9502, 2300 RA Leiden (The Netherlands) E-mail: a.kros@chem.leidenuniv. nl