Rational design and functionalization of circular tandem repeat proteins

项目摘要/摘要 自然进化产生了一系列令人惊叹的不同蛋白质，它们执行一套同样不同的 生物体中的分子功能。这些蛋白质--它们构成了 我们可能会寻求开发新的蛋白质试剂--在它们的突变历史过程中得到了磨练 以满足特定的功能挑战。因此，通过理性的手段将它们转变为新的功能通常 事实证明是有问题的：它们的表达和/或稳定性因我们的重新设计尝试而受到损害，并且它们的 遗迹功能与我们的预期用途不一致。全新的蛋白质设计，使用先进的计算机 识别稳定序列的算法：结构配对不依赖天然模板，可以产生蛋白质 在自然界中从未见过的折叠，因此为功能化提供了另一种蛋白质支架来源。 我们最近报道了一类特定蛋白质从头设计的新算法的开发- 环形串联重复蛋白或cTRPs-其模块化的、自我增强的对称结构提供 具有稳定性高、几何构型可调、低聚状态可切换等优点。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 下游应用的蛋白质支架来源。我们在这项建议中的目标是：第一，进一步发展 我们的算法是为了设计和实验验证一组不同大小和 第二，与哈钦森中心的临床同事合作，评估这些 设计作为脚手架，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分析中测试这些设计的结构，目标是加快 细胞疗法的发展。这项研究的成功完成将导致(1)蛋白质的改善 设计算法，该算法已在一系列拓扑中经过严格验证，并可用于 研究社区；(2)用于下游功能化的稳定和坚固的蛋白质支架家族，所有 其成员已被结构和生物物理表征；(3)一套有用的蛋白质试剂，用于 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.