BIOMIMETIC ASSEMBLIES

仿生组件

• 批准号: 8361121
• 负责人: MATTHEW TIRRELL
• 金额: $ 1.23万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361121
• 关键词: Alanine; Alkanes; Atomic Force Microscopy; Biological; Cell Death; Chemistry; Crowding; Data Analyses; Divalent Cations; Electron Microscopy; Funding; Grant; High Pressure Liquid Chromatography; Hydrophobic Interactions; Ice; Image; Ionic Strengths; Liquid substance; MDM2 gene; Membrane Lipids; Micelles; Morphologic artifacts; Morphology; National Center for Research Resources; Palmitic Acids; Peptides; Phase; Physiological; Preparation; Principal Investigator; Proteins; Research; Research Infrastructure; Resolution; Resources; Sampling; Shapes; Solid; Solutions; Source; Structure; Surface; System; Tail; United States National Institutes of Health; Water; aqueous; base; cancer cell; cost; cryogenics; inhibitor/antagonist; light scattering; macromolecule; research study; response; self assembly

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 肽两亲物 (PA) 因其自组装能力而成为一类极具吸引力的生物活性分子。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，由疏水核心和肽冠组成。有趣的是，肽折叠是对自组装的反应而发生的，很可能是由于拥挤效应。对组装和肽折叠的控制使材料能够以各种形态精确地显示功能。 我们制备了基于 p53_14-29 的肽两亲物，p53_14-29 是一种肽，可作为 p53-MDM2 相互作用的抑制剂，导致癌细胞死亡。我们的初步实验表明，向 p53_14-29 添加棕榈酸会导致两亲物形成拉长的胶束，结构明显扭曲。相反，在肽和疏水尾之间插入四个丙氨酸显示圆柱形胶束中没有扭曲。当向系统中添加二价阳离子时，差异更加明显：原子力显微镜证明，对于含有丙氨酸的 PA，分离的胶束发生快速聚集。另一方面，仅具有棕榈尾的样品表现出胶束的缓慢伸长，结构中存在明显的扭曲。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 TEM 不仅可以证实 AFM 观察结果，还可以提供有关所形成结构的更多细节。还有一个悬而未决的问题是扭曲结构的实际形状是什么：是扁平带还是扭曲圆柱体？ 我们最近还表明，由单尾烷烃形成的肽两亲胶束在生物液体和脂膜存在下不稳定。为了克服这个问题，合成了双尾PA。光散射研究表明各向异性散射体的形成。然而，数据分析需要形状信息，而这些信息可以通过玻璃冰中的 TEM 成像轻松获得。必须注意的是，由于自组装是由疏水效应驱动的，因此在样品制备过程中水的存在至关重要。 肽两亲物使用标准固相化学合成，并使用 HPLC 纯化以获得大于 95% 的纯度。这些 PA 的临界胶束浓度约为 1-5 uM。将使用至少高一个数量级的浓度来制备用于成像的溶液。光散射先前已表明生理离子强度不会改变胶束的大小，因此成像应在 PBS 10mM 中进行，无论有或没有二价阳离子。