Evolution of molecular complexes: genetic, structural, and functional mechanisms for the evolution of oligomers and allostery

我们建议对分子的历史起源和阐述进行首次实验研究。 复合体。几乎所有的蛋白质都与特定的分子伴侣组装成精确的几何形状 但我们对这些安排的遗传和结构机制知之甚少 复合体的进化或解释它们的起源、精化和长期的进化力量 坚持不懈。我们将把祖先蛋白质的重建与生化、结构和 重建三种模型蛋白质分子复合体演化的功能实验 家庭，使我们能够制定和测试关于进化原因和 化学计量、变构和专一性变化的后果。这三个蛋白质家族都是 生物学上基本的，实验上容易驯服的，并例证了不同的问题。这些模型包括：1) 血红蛋白，脊椎动物的主要氧气载体，是生物化学的标志性异构体 一个变构调节的分子复合体；2)柠檬酸合成酶，一种重要的代谢 酶在一些谱系中是二聚体，在另一些谱系中是变构调节的六聚体，这 提供了一个丰富的案例研究--关于变构和变构的进化关系和长期持久性 复杂性；以及3)类固醇激素受体，这是一个转录因子家族，调节 脊椎动物的繁殖和发育，并在基因复制后进化为特定的 组装成同源二聚体，每个具有不同的调节功能。该项目将解决这些问题 问题：1)络合物如何以及为什么在化学计量比中从简单形式产生和增加？ 2)什么遗传和生物物理机制调节了新的和特定的界面的进化？3)通过 变构进化的机制是什么？4)寡聚体相关功能的选择起作用了吗？ 完全是为了分子络合物的出现和长期存在？或者做了替换 只与组装的形式兼容的中立地发生，并巩固综合体，创造一个 进化朝着更复杂的方向发展？5)复制后，同分体是如何进化的 有选择地与自己的副本组装(不包括它们的姐妹鹦鹉)，这是进化 在实现这种选择性之前，有哪些新的功能受到限制？通过结合先进的技术 从蛋白质生物化学和进化生物学出发，该项目将在 关于进化力量和生化机制的史无前例的分辨率假说 是分子络合物的物理和功能性质的基础。这也将有助于解释 为什么多聚体如此广泛，并通过揭示进化如何实现专一性和变构 法规，加强工程工作，以设计具有这些特性的综合体。该项目将 也为今后其他分子络合物的研究提供了方法学和概念性模板。

We propose the first experimental studies of the historical origin and elaboration of molecular complexes. Virtually all proteins assemble with specific molecular partners into precise geometric arrangements, but we know little about the genetic and structural mechanisms by which these complexes evolved or the evolutionary forces that explain their origin, elaboration, and long-term persistence. We will combine ancestral protein reconstruction with biochemical, structural, and functional experiments to reconstruct the evolution of molecular complexes in three model protein families, enabling us to formulate and test general hypotheses about the evolutionary causes and consequences of changes in stoichiometry, allostery, and specificity. All three protein families are biologically essential, experimentally tractable, and exemplify distinct questions. The models are: 1) hemoglobin, the major oxygen carrier in vertebrates, a heterotetramer that is biochemistry's iconic case of an allosterically regulated molecular complex; 2) citrate synthase, an essential metabolic enzyme that is a dimer in some lineages and an allosterically regulated hexamer in others, which provides a rich case-study of the evolutionary relationship and long-term persistence of allostery and complexity; and 3) steroid hormone receptors, a family of transcription factors that regulate vertebrate reproduction and development and which evolved after gene duplication to specifically assemble as homodimers, each with distinct regulatory functions. The project will address these questions: 1) How and why do complexes originate and increase in stoichiometry from simpler forms? 2) What genetic and biophysical mechanisms mediate evolution of new and specific interfaces? 3) By what mechanisms did allostery evolve? 4) Does selection for oligomer-associated functions account entirely for the emergence and long-term persistence of molecular complexes? Or did substitutions compatible only with the assembled form occur neutrally and entrench the complex, creating an evolutionary ratchet towards greater complexity? And 5) After duplication, how did homomers evolve to selectively assemble with copies of themselves (excluding their sister paralogs), and was evolution of new functions constrained until this selectivity was achieved? By combining advanced techniques from protein biochemistry and evolutionary biology, the project will articulate and test at unprecedented resolution hypotheses about the evolutionary forces and biochemical mechanisms that underlie the physical and functional properties of molecular complexes. It will also help to explain why multimers are so widespread and, by revealing how evolution achieved specificity and allosteric regulation, enhance engineering efforts to design complexes with these properties. The project will also provide a methdological and conceptual template for future studies of other molecular complexes.