BIOMIMETIC ASSEMBLIES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 多肽两亲性(Pas)是一类具有自组装能力的生物活性分子。在水溶液中，疏水相互作用促使它们聚集成蛋白质类似结构，由疏水核心和多肽日冕组成。有趣的是，多肽折叠是在自我组装的情况下发生的，很可能是由于拥挤效应。对组装和多肽折叠的控制为材料提供了各种形态的功能的精确显示。 我们已经制备了基于P53_14-29的多肽两亲性，P53_14-29是一种抑制P53-MDM2相互作用的多肽，导致癌细胞死亡。我们的初步实验表明，将棕榈酸添加到P53_14-29上会导致两亲分子形成拉长的胶束，结构明显扭曲。相反，将四个丙氨酸插入多肽和疏水尾部之间，没有显示柱状胶束中的扭曲。当二价阳离子加入到体系中时，这种差异变得更加明显：原子力显微镜证明，对于含有丙氨酸的PA，孤立胶束发生了快速聚集。另一方面，只有掌状尾巴的样品显示胶束伸长缓慢，结构有明显的扭曲。尽管AFM成像是在液体中进行的，但平坦表面的存在可能与伪影有关，并且分辨率仅在一个方向上敏感。低温透射电子显微镜不仅可以证实原子力显微镜的观察结果，还可以提供有关形成结构的更多细节。还有一个问题仍然没有答案，那就是扭曲结构的实际形状是什么：它是扁平的丝带还是扭曲的圆柱体？ 我们最近还发现，由单尾烷形成的两亲多肽胶束在生物体液和脂膜存在下不稳定。为了克服这一问题，人们合成了双尾聚合物。光散射研究表明形成了各向异性的散射体。然而，对数据的分析需要形状信息，而这些信息很容易通过在玻璃冰中进行透射电子显微镜成像而获得。必须注意的是，由于自组装是由疏水效应驱动的，所以在样品制备过程中，水的存在是必不可少的。 用标准固相化学法合成了多肽两亲化合物，并用高效液相色谱法进行了纯化，得到了纯度大于95%的多肽两亲性化合物。这些PAS的临界胶束浓度在1-5um量级。将使用至少一个数量级更高的浓度来准备成像溶液。光散射以前已经表明，生理离子强度不会改变胶束的大小，因此成像应在PbS 10 mm中进行，无论是否有二价阳离子。

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.