Computational design of specific binding proteins using Leave-One-Out

描述（由申请人提供）：本研究的目标是能够为任何蛋白质靶标设计受体蛋白。此外，我们提出结合事件将通过酶活性或荧光的出现来信号化。这种新型结合蛋白将能够感知并报告其他混合物中特定蛋白质或肽的存在，从而在体内和体外检测疾病因子或其他感兴趣的蛋白质。这种新方法利用了蛋白质折叠途径。当蛋白质折叠时，它们按照特定的事件顺序进行，这些事件可以根据蛋白质的结构来预测。当蛋白质完成折叠时，它的活性立即被激活。如果我们遗漏了一小段蛋白质，使折叠无法完成，那么蛋白质就会处于非活性状态，直到缺失的那部分出现。使用这种“留一个”策略，部分折叠的蛋白质就会成为缺失片段的传感器。利用计算设计算法，一个新的氨基酸序列可以取代被遗漏的片段。这个新的序列可以来自炭疽、禽流感、癌细胞标记物或任何其他蛋白质。利用大规模并行计算集群，并根据已知的蛋白质折叠和能量计算，将设计任务划分为许多较小的任务，从而使围绕该新序列的氨基酸的计算替代成为可能。最终的设计是一种包裹在目标肽周围的蛋白质，通过它的互补形状来明确识别它。绿色荧光蛋白一直是第一个“留一设计”研究的主题，并产生了特定的结合蛋白，仅当目标肽存在时才发光。第一个目标是从致命的H5N1禽流感毒株中提取的肽。当设计的Leave-One-Out GFP遇到它的目标肽时，它完成折叠并发出荧光。

DESCRIPTION (provided by applicant): The goal of this research is to be able to design a receptor protein for any protein target. Furthermore, we propose that the binding event will be signaled by the appearance of enzyme activity or fluorescence. The novel binding proteins will be able to sense and report the presence of a specific protein or peptide in a mixture of others, allowing the detection of disease agents or other proteins of interest both in vivo and in vitro. The new approach takes advantage of protein folding pathways. When proteins fold, they do so in a specified order of events, and the events can be predicted based on the structure of the protein. When a protein finishes folding, its activity is immediately turned on. If we leave out one small piece of the protein so that the folding cannot finish, then the protein sits in an inactive state until the missing piece appears. Using this Leave-One- Out strategy, partially folded proteins become sensors for their missing pieces. Using computational design algorithms, a new amino acid sequence can be substituted for the left out piece. This new sequence can be from anthrax, avian flu, a cancer cell marker or any other protein. Computational substitution of the amino acids surrounding this new sequence is made possible using massively parallel computing clusters, and by dividing the design task into numerous smaller tasks based on what is known about protein folding and energy calculations. The final design is a protein that wraps around the target peptide, specifically identifying it by its complementary shape. Green fluorescent protein has been the subject of the first leave-one-out design studies and has yielded specific binding proteins that glow only when the target peptide is present. The first target was a peptide from the deadly H5N1 strain of avian flu. When the designed Leave-One-Out GFP encounters its target peptide, it finishes folding and becomes fluorescent. PUBLIC HEALTH RELEVANCE: The results of this research could revolutionize protein diagnostics, replacing monoclonal antibodies as the current best means of specific protein identification. Computationally designed specific binding proteins could be used as protein therapeutics, biosensors, proteomic arrays, fluorescent probes, protein purification affinity agents, and many other applications.