| Here we describe a general approach to de novo design of self assembling peptides based on the alpha-helical coiled coil motif derived from the GCN4 transcription factor. Peptides of 41--44 amino acids, defining six distinct heptad repeats, as well as their derivatives, were prepared via conventional SPPS with minor condition modifications. Buried polar aminoacid interactions in the hydrophobic core and electrostatic interactions of residues in positions e/g were used to direct the registry of the neighboring alpha-helices within self propagating coiled coil structures.; The paradigm was first explored in a homodimeric system S1, which self-assembly was examined at varying levels of structural hierarchy for compliance of the observed structures with the experimental model. CD spectroscopy provided evidence for an alpha-helical coiled coil structure for S1 in aqueous solution, which could be reversibly denatured through thermal methods. TEM measurements indicated the formation of long aspect-ratio fibers of uniform diameter from aqueous solutions of S1 , however the dimensions of the fibers suggested the existence of lateral association between the fibrils corresponding to the 2-stranded helical bundles. The alpha-helical coiled coil structure was confirmed in the solid-state for fibers derived from self-assembly of S1 by a combination of wide-angle X-ray diffraction, 13C CP/MAS and 13C{lcub} 15N{rcub} REDOR SS NMR spectroscopy. Small-angle neutron scattering (SANS) and synchrotron small-angle X-ray scattering (SAXS) measurements on diluted aqueous solutions of S1 provided a fibril diameter that corresponded to the lateral dimensions estimated for a dimeric coiled coil assembly on the basis of structural determinations of model peptides.; Several homotrimeric self assembling peptides, created using similar principles, were also shown to be highly alpha-helical and fibrillogenic. The most studied homotrimeric system, S6K, was modified with biotin-containing tethers to give the forming fibers ability to direct secondary assembly of exogenous avidin-conjugated objects on the fibers' surface, which was confirmed by TEM studies of arranged ExtrAvidin gold and fluorescence studies of streptavidin-conjugated CdSe nanoparticles.; Patterned incorporation of Histidine residues into an every other heptad of the S6K's highly hydrophobic isoleucine core leads to noticeable decrease of stability. More importantly, clustered imidazole side chains were shown to modulate fibrillogenicity, with the protonated form, existing below pH 5.8, completely unable to form continuous ordered assemblies, as shown by CD, microscopy and microrheology. We hypothesize that the main reason of the slight difference between the observed pH limit of 5.8 and pKa of a Histidine side chain is the effect of imidazole groups' hydrophobic shielding by the Isoleucine residues.; Derived principles could be applied towards creation of novel biomaterials, sensors, drug delivery systems and nano-scaled arrays. |
