Vectorial Folding of Large, Multidomain Proteins

标题：大型多结构域蛋白质的矢量折叠蛋白质是由连接成聚合物样链的氨基酸构建块组成的生物分子。它们在生命过程中至关重要，因为它们为细胞提供了结构元素，并发挥了许多活跃的细胞功能，例如在红细胞中作为氧载体，在肌肉中产生收缩力，并因为它们能够识别和结合外来分子而支持免疫系统。它们执行细胞功能的能力取决于蛋白质折叠成适当的结构。当蛋白质错误折叠时，它们不仅会失去功能，还会导致阿尔茨海默氏症和帕金森病等疾病。迄今为止，在理解小的（约100个氨基酸）模型蛋白如何获得三维结构方面取得了重大进展，然而，对主导所有生命形式的大的、复杂的、多结构域的蛋白质的折叠机制知之甚少。该项目的长期目标是通过实验和计算技术的结合，促进对大型、复杂、多结构域蛋白质折叠机制的理解。该项目将为3名研究生和6名本科生提供一个令人兴奋的跨学科教育和研究机会。该项目还结合了几个实验学科，它将为所有参与人员，初级和高级研究人员提供丰富的学习经验。拓展活动将包括K12学生和他们的老师。在这个项目中，大型，复杂，多结构域蛋白质的折叠机制将使用实验和计算技术的结合进行检查。大的多结构域蛋白质在矢量约束条件下顺序折叠以避免错误折叠的假设，而不是小蛋白质典型的两态方式，将被检查。单分子蛋白质折叠数据将通过DNA和蛋白质工程与基于原子力显微镜和光阱的单分子力谱（AFM-SMFS, OT-SMFS）测量相结合来获得。计算方法，如操纵分子动力学（SMD）将与实验测量并行执行，并有助于解释数据。多结构域蛋白在解释AFM-SMFS和OT-SMFS数据时存在一个独特的问题：产生信号的结构域必须从蛋白质的多个结构域中识别出来。为了避免这个问题，新型的多肽“折叠探针”，如反平行卷曲线圈，将被开发出来，可以插入到大蛋白质的关键位置，当SMFS询问时，报告周围结构的折叠状态。这些研究将研究一些大型蛋白质的机械折叠，这些蛋白质的大小和结构复杂性不断增加，支持它们重要的生物学功能，如ATP合成或DNA复制。这项研究将促进对生物学中最基本的未解决问题之一的认识；允许长氨基酸链折叠成复杂的多结构域结构的机制。

Title: Vectorial Folding of Large, Multidomain Proteins Proteins are biological molecules composed of amino acid building blocks connected into polymer-like chains. They are critically important in life processes by providing structural elements to cells and by performing many active cellular functions, for example as oxygen carriers in red blood cells, by generating contractile forces in muscles and by supporting the immune system because they are able to recognize and bind foreign molecules. Their ability to perform their cellular functions is dependent upon proteins folding into the appropriate structure. When proteins misfold, not only do they loose function but they can also cause diseases such as Alzheimer's and Parkinson's disease. To date, significant progress has been made toward understanding of how small (~100 amino acids), model proteins acquire their three-dimensional structures, however, very little is known about folding mechanisms of large, complex, multi-domain proteins that dominate all forms of life. The long-term objective of this project is to advance understanding of the folding mechanisms of large, complex, multi-domain proteins by using a combination of experimental and computational techniques. This project will provide an exciting interdisciplinary education and research opportunity for 3 graduate and 6 undergraduate students. This project also combines several experimental disciplines and it will provide a rich learning experience for all people involved, junior and senior researchers alike. Outreach activities will involve K12 students and their teachers.In this project the mechanism of folding of large, complex, multi-domain proteins will be examines using a combination of experimental and computational techniques. The hypothesis that large multi-domain proteins fold sequentially under vectorial constraint conditions to avoid misfolding, and not in a two-state-fashion typical of small proteins, will be examined. Single-molecule protein folding data will be acquired by combining DNA and protein engineering with Atomic Force Microscopy-based and optical trap-based single-molecule force spectroscopy (AFM-SMFS, OT-SMFS) measurements. Computational methods such as Steered Molecular Dynamics (SMD) will be performed in parallel to experimental measurements and aid in interpreting data. Multi-domain proteins present a unique problem for interpreting AFM-SMFS and OT-SMFS data: the domain that gives rise to a signal must be identified out of the multiple domains in the protein. To circumvent this issue, novel polypeptide-based 'folding probes' such as antiparallel coiled-coils will be developed that can be inserted into large proteins at critical positions to report on the folding status of the surrounding structure when interrogated by SMFS. These studies will examine mechanical folding of a number of large proteins with increasing size and structural complexity that supports their important biological functions such as ATP synthesis or DNA replication. The research will advance the knowledge about one of the most fundamental unsolved problems in biology; mechanisms allowing long amino acid chains to fold into complex, multidomain structures.