Proceedings of the 4th International Peptide Symposium in conjunction with the 7th Australian Peptide Conference and the 2nd Asia-Pacific International Peptide Symposium, 2007
Jackie Wilce (Editor) on behalf of the Australian Peptide Association
Understanding self-assembled amphiphilic peptide supramolecular structures from primary sequence helix propensity. M. Baumann, E. Reimhult, M. Textor Department of Materials Science, Laboratory for Surface Science and Technology (LSST), ETH Zürich, Zurich, Switzerland E-mail:
[email protected]
Introduction Peptides are attracting increasing interest as components to build engineered biological materials, e.g., for cell culture substrates, tissue engineering scaffolds and drug carrier systems targeting specific compartments of the body. Selected peptides and hybrid peptides containing aliphatic hydrocarbon moieties have been shown to self-assemble, under appropriate conditions, into a variety of three-dimensional, supramolecular structures with micelles [1,2], vesicles [3] nano scale fibers [4-6] and fiber network scaffolds [5,7,8] as structural motives. These structures provide means of mimicking physiological functions such as ion transport through cell membranes, structural components of cells as well as model certain diseases caused by e.g. amyloids [9]. Furthermore, interactions between peptides and lipid membranes regulate a wide range of biological phenomena, including the translocation of polypeptides through membranes and the cytosolic action of antimicrobial peptides. Recent work by Ghadiri and coworkers has shown, that designed peptide nanotubes can insert in lipid bilayers and allow ion transfer through them [10,11]. Designing and functionalizing structures on the nanoscale is a goal of research fields ranging from materials science to nanomedicine [12]. Self-assembly of molecular building blocks presents a promising route towards controlled engineering of functional macromolecular parts. Another advantage of using a self-assembling system is that structures can be built in a parallel process. The ability to self-assemble into ordered structures as well as the straightforward production of peptides makes them excellent building blocks to design and build biocompatible structures on the nanoscale, with relevant applications in e.g. drug delivery vehicles, tissue engineering scaffolds and nano-templating [13,14]. To gain control over the formed structures, insight into the physicochemical processes underlying the self-assembly is crucial. We investigate the influence of systematic changes in the amino acid sequence of short amphiphilic peptides on the self-assembled macromolecular structures. By varying only one parameter (e.g. helix propensity) between the investigated peptides the direct effect on the macromolecular structures can be tied to that property of the primary sequence of the peptide monomers. Cationic surfactant-like peptides with systematically varied tail regions serve in this study as model peptides. Such peptides are especially of interest for investigating interactions with negatively charged cell membranes, e.g. for drug delivery applications. Zhang and coworkers showed that such peptides, resembling phospholipids from
biological membranes in dimension and architecture, undergo self-assembly in aqueous solution to form tubular structures [14]. Two positively charged amino acids comprise the polar head, and the hydrophobic tail is formed by a repetitive sequence of one non-polar amino acid. Valine, leucine and isoleucine were chosen as tail forming hydrophobic amino acids, since they share a similar overall hydrophobicity [15] but differ in helix propensity ( valine < isoleucine < leucine) [16] (Figure 1). Helix propensity, due to different branching modes of the amino acid side chains, is an interesting parameter to vary since different secondary structure preferences of the monomers should lead to varied sterical demands of the monomers in the assembled structures and therefore to varieties in the macromolecular shape.
Fig. 1 Helix propensity values vs. hydrophobicity of selected amino acids. The aliphatic amino acids valine 15 isoleucine and leucine share a similar hydrophobicity 16 while differing in helix propensity . Among the charged amino acids which were considered to form the head group lysine was chosen because it is only bearing one heteroatom (N) in the side chain to give one positive charge per molecule below pH 9.21.
Results and Discussion The self-assembled structures of Ac-Ile6Lys2-NH2 (I6K2), Ac-Leu6Lys2-NH2 (L6K2) and Ac-Val6Lys2-NH2 (V6K2) were characterized as function of concentration and temperature in a low salt aqueous solution. For I6K2 different ionic strengths of the aqueous solution were tested as well. The peptide solutions were 1
Jackie Wilce (Editor) on behalf of the Australian Peptide Association
Proceedings of the 4th International Peptide Symposium in conjunction with the 7th Australian Peptide Conference and the 2nd Asia-Pacific International Peptide Symposium, 2007 incubated for 24 h at 4°C prior characterization. Characterizations of the assembled supramolecular structures were made using transmission electron microscopy (TEM). 400 mesh copper grids were coated with a 10 nm carbon film and treated with air plasma prior to sample adsorption. After washing the specimens with ultra pure water they were negative stained with 1 % uranyl acetate. Atomic force microscopy (AFM) in tapping mode was used to asses the structure height and confirm the TEM micrographs. Samples were adsorbed on freshly cleaved mica. After washing the samples were air dried prior to examination. To probe the secondary structure of the peptides in the assembly circular dichroism (CD) spectra were measured. For all three peptides distinct macromolecular structures were found with TEM and AFM. I6K2 assembles into flat ribbon-like structures (fig 2 (a)). When comparing pure MilliQ water as solvent to 2 mM sodium chloride solution an increase in diameter of the tubules can be observed, sometimes even adding up to small sheet-like structures (fig.2(b)). The height determined with the AFM in air after drying of incubated peptide solution corresponds roughly to the length of two monomers (3.5 - 4.0 nm).
with large random coil contents are not increasingly ordered by the same process. The detailed effect of heat annealing on the assembled structure remains to be determined.
Acknowledgments Swiss National Science Foundation, NCCR project “Nanoscale Science” for financial support.
References 1. 2.
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Fig. 2 Influence of ionic strength on sheet width and length of I6K2. Micellular tubules are formed when I6K2 is dissolved in MilliQ (a). Sheet-like, broader structures are observed when I6K2 is dissolved in 2 mM NaCl solution (b).
CD reveals a β-sheet structure for I6K2 peptides in the assemblies. Upon increasing the temperature for 2 h after preparing the peptide solutions an increase in secondary structure order for the I6K2 assemblies could be observed. The higher the temperature selected during the assembly the stronger was the CD β-sheet signal as verified for temperature cycles up to 90 °C. For both L6K2 and V6K2 the CD signal revealed a random coil structure with α-helical content. L6K2 and V6K2 monomers assemble into mostly micellar fibrils depending on peptide concentration, as verified by TEM and AFM. No change was observed after temperature increase during the incubation time for the secondary structure in the macromolecular aggregates of either V6K2 or L6K2. In summary, we have demonstrated for a selection of designed amphiphilic peptides that a peptide primary structure yielding β-sheet secondary structure assembles into a more sheet-like superstructure than peptides coding for more random coil/α-helical secondary structure. The latter predominantly results in micellar fibrilar assemblies. Furthermore, while the β-sheet secondary structures can be annealed by a high temperature cycle α-helical structures
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