This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Peptide amphiphiles (PAs) are an attractive class of bioactive molecules due to their self-assembling capabilities. In aqueous solutions, hydrophobic interactions drive their aggregation into protein analogous structures, composed of a hydrophobic core and a peptide corona. Interestingly, peptide folding occurs in response to self-assembly, most likely due to crowding effects. Control over assembly and peptide folding provides materials with precise display of functionalities, in a variety of morphologies. We have prepared peptide amphiphiles based on p53_14-29, a peptide that acts as an inhibitor of the p53-MDM2 interaction, resulting in cancer cell death. Our initial experiments have shown that addition of palmitic acid to p53_14-29 resulted in amphiphiles that formed elongated micelles, with an apparent twist in the structure. In contrast, insertion of four alanines between the peptide and the hydrophobic tail showed no twisting in the cylindrical micelles. When divalent cations were added to the system, the differences were accentuated: for the PA with the alanines, rapid aggregation of isolated micelles occurred, as evidenced by atomic force microscopy. On the other hand, the sample with only the palmitic tail showed a slow elongation of micelles with a clear twist in the structure. Even though AFM imaging took place in liquid, the presence of a flat surface might be associated with artifacts and the resolution is sensitive only on one direction. Cryogenic TEM would not only confirm AFM observations but also provide with more details on the formed structures. A question that also remains unanswered is what is the actual shape of the twisted structures: is it a flat ribbon or a twisted cylinder? We have also recently showed that peptide amphiphile micelles formed by single-tail alkanes are not stable in presence of biological fluids and lipid membranes. In order to overcome this problem, double-tailed PAs have been synthesized. Light scattering studies indicate formation of anisotropic scatterers. However, analysis of the data requires shape information, which would readily be available through TEM imaging in vitreous ice. It must be noted that since self-assembly is driven by the hydrophobic effect, the presence of water is essential during sample preparation. Peptide amphiphiles are synthesized using standard solid phase chemistry and purified using HPLC to attain purities greater than 95 %. The critical micelle concentration of these PAs is in the order of 1-5 uM. At least an order of magnitude higher concentration will be used to prepare solutions for imaging. Light scattering has previously shown that physiological ionic strength does not alter size of micelles and therefore imaging should be performed in PBS 10mM, with or without divalent cations.
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