Rational design and functionalization of circular tandem repeat proteins

项目总结/摘要 自然进化已经产生了一系列惊人的多样化的蛋白质，它们执行着同样多样化的功能。 生物体中的分子功能。这些蛋白质构成了 我们可能会寻求开发新的蛋白质试剂-在它们的突变历史中已经磨练过了 以应对特定的功能挑战。因此，通过合理的手段将它们转换为新的功能， 证明是有问题的：它们的表达和/或稳定性受到我们重新设计尝试的影响， 遗迹功能与我们的预期用途不一致从头蛋白质设计，它使用复杂的计算机 识别稳定序列的算法：不依赖于天然模板的结构配对，可以创建蛋白质 折叠从未在自然界中见过，因此提供了功能化蛋白质支架的替代来源。 我们最近报道了一种新算法的发展，用于从头设计一类特定的蛋白质- 环状串联重复蛋白或cTRPs-其模块化的，自我增强的对称结构提供了 这些优点包括高稳定性、可调几何形状和可转换的低聚状态。我们假设 一般来说，从头设计的蛋白质，特别是这些设计的cTRP，将被证明是一个有价值的 下游应用蛋白质支架的来源。我们提出这一建议的目的是，首先，进一步发展 我们的算法，以设计和实验验证一组不同大小的cTRP支架， 第二，与哈钦森中心的临床同事合作，评估这些 设计作为支架用于呈现具有精确控制的对称性和几何形状的功能域。 我们的合作者将在细胞测定中测试这些设计的构建体，目的是加速细胞的生长。 细胞疗法的发展。这项研究的成功完成将导致（1）改善蛋白质 设计算法，这些算法已在一系列拓扑结构中得到严格验证， 研究社区;（2）稳定和强大的蛋白质支架家族，用于下游功能化，所有 其成员已在结构上和生物药理学上表征;（3）一组有用的蛋白试剂， 生物医学应用

PROJECT SUMMARY/ABSTRACT Natural evolution has produced a stunningly diverse array of proteins that perform an equally diverse set of molecular functions in living organisms. These proteins—which constitute the primary raw material from which we might seek to develop new protein reagents—have been honed over the course of their mutational history to meet specific functional challenges. As a result, turning them to new functions by rational means often proves problematic: their expression and/or stability are compromised by our reengineering attempts, and their relic functionality is at odds with our intended use. De novo protein design, which uses sophisticated computer algorithms to identify stable sequence:structure pairings without relying on native templates, can create protein folds never before seen in Nature, and thus offers an alternative source of protein scaffolds for functionalization. We recently reported the development of new algorithms for de novo design of a particular class of proteins— circular tandem repeat proteins or cTRPs—whose modular, self-reinforcing symmetrical architecture offers advantages that include high stability, tunable geometry, and switchable oligomeric state. We hypothesize that de novo designed proteins in general, and these designed cTRPs in particular, will prove to be a valuable source of protein scaffolds for downstream application. Our aims in this proposal are first, to further develop our algorithms in order to design and experimentally validate a diverse set of cTRP scaffolds of varied size and topology; and second, in collaboration with clinical colleagues here at the Hutchinson Center, to evaluate these designs as scaffolds for presentation of functional domains with precisely controlled symmetry and geometry. Our collaborators will test these designed constructs in cellular assays with the goal of speeding the development of cellular therapies. Successful completion of this research will lead to (1) improved protein design algorithms that have been rigorously validated across a range of topologies and are available to the research community; (2) a family of stable and robust protein scaffolds for downstream functionalization, all of whose members have been structurally and biophysically characterized; (3) a set of useful protein reagents for biomedical applications.