Control and functionalization of protein nanofibrils using genetic and chemical modification to define assembly mechanisms and design principles

该研究项目旨在更好地了解蛋白质聚集在食品质量，安全和人类健康中的应用。蛋白质淀粉样原纤维或“纳米原纤维”是与疾病相关的高度组织化类型的蛋白质聚集体（例如，阿尔茨海默氏症，ALS和疯牛病），而在其他情况下，他们发挥功能性作用（例如，细菌生物膜、藤壶粘附和人类黑色素合成）。对蛋白质纳米原纤维形成的机理理解可以用于防止不希望的聚集（即，疾病或细菌生物膜形成），并开发新型仿生材料和装置，如催化阵列、生物传感器、细胞支架、生物电子学和生物活性化合物的控释。这些可以应用于食品加工（生物反应器和生物传感器的酶固定化），组织工程（用于食品的培养肉类;用于医学的培养组织）或作为功能性食品（生物活性物质的受控释放）。蛋白质纳米原纤维具有几种独特的性质，包括高纵横比（=1000：10 nm）、强度和稳定性、独特的表面化学和自繁殖能力。 一个主要的挑战是构建蛋白质纳米纤维的设计规则并不为人所知。蛋白质可以被诱导自组装成纳米原纤维，即几个分子宽和1000个分子长的蛋白质线，这是一个复杂的过程，该过程遵循不同的途径，包括蛋白质解折叠、错误折叠、寡聚体形成、原纤维和成熟原纤维形成。第二个挑战是利用蛋白质纳米原纤维来产生功能材料需要能够以与原纤维结构相容的方式对蛋白质进行位点特异性、选择性地化学修饰。靶向蛋白质上的特定位置通常不能依赖于天然丰富的功能团（例如，胺、羧酸和巯基），因为这些存在于多个位置并且它们的反应性限于某些pH和氧化还原条件。 为了克服这些局限性，将使用新的蛋白质工程工具在基因上将“点击”化学基团插入模型蛋白质的特定位点，以便以明确、有效的方式进行化学标记。这些点击基团对生物分子是惰性的，并且仅与特定的配偶体反应，其将用于将纳米原纤维与其他功能分子（例如，肽、酶、碳水化合物、脂质、DNA、纳米颗粒、荧光染料）。生物物理工具将被用来检查这种遗传和化学修饰对原纤维组装，结构和功能的影响。该平台将用于研究原纤维组装的机制，并创建新的纳米级材料和设备（例如，生物传感器，有序的酶阵列和受控释放材料），最终可能有助于提高食品质量和安全。

This research program seeks a better understanding of protein aggregation for applications in food quality, safety and human health. Protein amyloid fibrils, or `nanofibrils', are a highly organized type of protein aggregate associated with disease (e.g., Alzheimer's, ALS & mad cow) in certain cases, while in other cases they play a functional role (e.g., bacterial biofilm, barnacle adhesion, and human melanin synthesis). A mechanistic understanding of protein nanofibril formation could be used both to prevent unwanted aggregation (i.e., disease or bacterial biofilm formation) and to develop novel biomimetic materials and devices, such as catalytic arrays, biosensors, cell-scaffolds, bioelectronics, and controlled-release of bioactive compounds. These may find applications in food processing (enzyme immobilization for bioreactors and biosensors), tissue engineering (cultured meat for food; cultured tissues for medicine) or as functional foods (controlled release of bioactives). Protein nanofibrils have several unique properties, including a high aspect ratio (=1000:10 nm), strength, and stability, a unique surface chemistry, and the ability to self-propagate. A major challenge is that the design rules for building protein nanofibrils are not well known. Proteins can be induced to self-assemble into nanofibrils, threads of protein a few molecules in width and 1000's in length, in a complex process that follows different pathways, involving protein unfolding, misfolding, oligomer formation, proto-fibril and mature fibril formation. A second challenge is that utilization of protein nanofibrils for creating functional materials requires the ability to chemically modify proteins site-specifically, selectively, and in a manner compatible with the fibril structure. Targeting specific locations on a protein generally cannot rely on naturally abundant functional groups (e.g., amine, carboxylic acid and sulfhydryl groups), as these are present at multiple locations and their reactivity is limited to certain pH and redox conditions. To overcome these limitations, new protein engineering tools will be used to genetically insert `click' chemical groups into specific sites of model proteins to allow for chemical labelling in a well-defined, efficient manner. These click groups are inert to biological molecules and react only with a specific partner, which will be used to conjugate the nanofibrils with other functional molecules (e.g., peptides, enzymes, carbohydrates, lipids, DNA, nanoparticles, fluorescent dyes). Biophysical tools will be used to examine the effects of such genetic and chemical modifications on fibril assembly, structure and functionality. This platform will be used to examine the mechanism of fibril assembly and to create new nanoscale materials and devices (e.g., biosensors, ordered enzyme-arrays, & materials for controlled release) that ultimately may help enhance food quality and safety.