A System of Minimalist Protein Labels for Fluorescence Studies

这个项目是由化学部的生命过程化学计划和分子和细胞生物科学部的分子生物物理学簇共同资助的。生命科学化学计划和分子生物物理学簇资助宾夕法尼亚大学的E.James Petersson教授开发工具来研究蛋白质运动的分子细节。为了发挥功能，蛋白质必须折叠成正确的形状，并具有适当的灵活性。动态的形状对健康的细胞很重要，但折叠成错误的形状会导致蛋白质对细胞产生毒性。彼得森实验室正在开发一套计算和实验工具系统来研究蛋白质形状的变化。这些工具由荧光标记组成，当蛋白质在细胞中制造时，这些标记可以同时并入。此外，正在开发预测最佳标记位置的计算方法。这些计算工具还帮助产生和解释结果，这些结果可以用来制作蛋白质运动的电影，这将有助于更好地理解动态环境中的蛋白质形状。重要的是，标签材料和计算机程序都可以通过网站向其他实验室提供。这项工作的更广泛影响包括在多学科实验室环境中培训本科生和研究生。训练环境利用有机合成、物理化学以及分子和细胞生物学来研究生物现象。该项目还在开发动手演示，向K-12学生传授荧光和蛋白质折叠在健康和疾病方面的知识。为了使这些受欢迎的活动更容易获得，正在提供视频和可下载的材料。这些材料允许K-12教师与他们的学生进行类似的练习。建议的研究产品和教育工具都是为使广大受众受益而设计的。蛋白质的灵活性使他们能够接触到各种折叠结构。这些结构之间的转换被用来在细胞内传递信号，促进酶催化，甚至在马达蛋白的情况下产生机械力。最近，人们的注意力集中在内在无序的蛋白质上。这些蛋白质没有稳定的结构，但在细胞健康和疾病中发挥着重要的功能作用。彼得森实验室正在开发基于芳香小支架的新型荧光氨基酸探针。这些探针在基因上被结合到多肽骨架中，对蛋白质折叠的干扰最小。该项目建立在首席研究员的发现基础上，即肽骨架的硫代酰胺取代可以作为小的荧光猝灭剂。在这种情况下，产生了同样最低限度的侧链硫代酰胺探针并对其进行了遗传编码。荧光氨基酸和/或硫代酰胺猝灭剂的光物理研究被用来将它们配对成适当的组。这些对被用来探测蛋白质中所有相关距离范围内的构象变化。这些最低限度的工具在共翻译蛋白质折叠研究或标记折叠蛋白质的内部方面特别有价值。这样的实验目前还不能用普通的翻译后荧光标记策略进行。该项目还在创建计算工具，以确定标签合并的非干扰位置。一种将荧光数据作为构象约束纳入蛋白质运动模型的策略正在开发中。这些计算工具是在流行的开源平台Rosetta上实现的，因此蛋白质研究社区可以访问和修改它们。

This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.The Chemistry of Life Sciences Program and the Molecular Biophysics Cluster fund Professor E. James Petersson of the University of Pennsylvania to develop tools to study the molecular details of protein motions. In order to function, proteins must fold into the correct shape and with the proper flexibility. Dynamic shapes are important to healthy cells, but folding into the wrong shape can cause a protein to become toxic to cells. The Petersson laboratory is developing a system of computational and experimental tools to study protein shape changes. The tools consist of fluorescent labels that can be incorporated concomitantly as proteins are made in cells. In addition, computational methods for predicting the best labeling sites are being developed. These computational tools are also assisting in producing and interpreting results that can be used to make movies of protein motions, which will be helpful to better understand protein shape in a dynamic environment. Importantly, both the labeling materials and computer programs are being made available to other laboratories via websites. The broader impacts of this work include training undergraduate and graduate students in a multidisciplinary laboratory environment. The training environment makes use of organic synthesis, physical chemistry as well as molecular and cellular biology to investigate biological phenomena. The project is also developing hands-on demonstrations to teach K-12 students about fluorescence and protein folding in health and disease. In order to make these popular activities more accessible, videos and downloadable materials are being made available. These materials allow K-12 teachers to carry out similar exercises with their students. Both the proposed research products and educational tools are designed to benefit a wide audience.The flexibility of proteins allows them to access a variety of folded structures. Shifts between these structures are used to transmit signals within cells, facilitate enzymatic catalysis, or even generate mechanical force in the case of motor proteins. Recently, much attention has focused on intrinsically disordered proteins. These proteins have no stable structures and yet play important functional roles in cell health and disease. The Petersson Laboratory is developing new fluorescent amino acids probes based upon small aromatic scaffolds. These probes are genetically incorporated into the peptide backbone and are minimally perturbing to protein folding. The project builds upon the principal investigator's discovery that thioamide substitutions of the peptide backbone can serve as small fluorescence quenchers. In this case, equally minimalist sidechain thioamide probes are generated and genetically encoded. Photophysical studies of the fluorescent amino acids and/or thioamide quenchers are used to pair them in appropriate sets. The pairs are used to probe conformational changes over all relevant distance ranges in proteins. These minimalist tools are particularly valuable in co-translational protein folding studies or labeling the interior of a folded protein. Such experiments are currently inaccessible to common post-translational fluorescent labeling strategies. The project is also creating computational tools to identify non-perturbing sites for label incorporation. A strategy for incorporating fluorescence data as conformational constraints in models of protein motion is being developed. The computational tools are implemented in the popular, open-source platform Rosetta, so that they can be accessed and modified by the protein research community.