Orthogonal Crosslinking for Protein Stabilization and Cytosolic Delivery

大量的蛋白质可以很容易地从细菌中产生，因此，代表了生物催化剂和治疗的可持续来源。凭借这一奖项，化学学部的生命过程化学项目资助了纽约州立大学布法罗分校的林清博士，以开发一种对蛋白质进行化学修饰的方法，当与光一起使用时，可以提高蛋白质的稳定性和进入细胞的能力。这种化学生物学策略使基于蛋白质的催化剂的设计具有更高的耐热性和化学物质的结构破坏能力。此外，这种新的修饰使从抗体（称为纳米体）衍生的细胞穿透小蛋白质得以发展，用于疾病的诊断和治疗。该项目通过与纽约州大学科学技术入学计划合作，为本科生提供跨学科研究机会，包括一些在STEM领域代表性不足的群体。2-芳基-5-羧基四唑（ACT）连接氨基酸与蛋白质的位点特异性结合，允许通过与近端亲核残基的分子内光交联产生首尾相连的环状蛋白质。本项目还探索了基于act的正交链间交联，用于合成具有正电荷表面的纳米体，以增强细胞穿透性。通过化学合成优化了各种结构体的交联效率和选择性。正交交联对蛋白质构象稳定性和细胞质摄取的影响用适当的生物测定方法进行了评估。这些研究有望对蛋白质内部光生成活性物质的反应性产生新的见解，从而设计出具有新结构和功能的蛋白质。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Large quantities of proteins can readily be produced from bacteria and, therefore, represent a sustainable source of biological catalysts and therapeutics. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Qing Lin from the State University of New York at Buffalo to develop a chemical modification to proteins that, when used with light, increases their stability and their ability to enter cells. This chemical biology strategy enables the design of protein-based catalysts with increased resistance to structural disruptions from heat and chemicals. In addition, this new modification enables the development of cell-penetrating small proteins derived from antibodies (called nanobodies) for the diagnosis and treatment of disease. The project provides interdisciplinary research opportunities to undergraduate students, including some from the groups underrepresented in STEM fields, through collaborations with the New York State Collegiate Science and Technology Entry Program.Site-specific incorporation of a 2-aryl-5-carboxytetrazole (ACT) linked amino acid into a protein allows the creation of head-to-tail circular proteins through intramolecular photo-crosslinking with a proximal nucleophilic residue. ACT-based orthogonal inter-strand crosslinking is also explored in this project for the synthesis of nanobodies that display positively charged surfaces to enhance cell-penetration. The crosslinking efficiency and selectivity of the various constructs are optimized through chemical synthesis. The effects of orthogonal crosslinking on protein conformational stability and cytosolic uptake are assessed using appropriate biological assays. These studies are expected to yield new insights into the reactivity of photo-generated reactive species inside proteins, which leads to designed proteins with novel structures and functions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.