Mutational Pleiotropy, Epistasis, and the Adaptive Evolution of Hemoglobin Function

新表型的逐步进化是生物学中几个基本问题的核心。在……里面 随着对新蛋白质功能的研究，这个问题在实验上变得容易解决，如果能够识别和 从功能上描述一整套致病突变。有了这样的系统，就可以解决 关键问题：新的功能是否通过每个功能产生的有益突变的连续固定而进化 当它们第一次出现时，表型的适应性变化？或者，蛋白质的进化转变 中性突变促进的功能，当它们第一次出现时不会产生适应性益处，但 增强后续突变对功能改变的影响？通过重建所有可能的突变 连接祖先和后代蛋白质的途径也有可能解决基本问题 关于偶然性和决定论在蛋白质进化中的作用。例如：新功能可以进化吗？ 从任何可能的祖先起点出发，或者特定的进化结果取决于先前的历史？ 我们将通过实验解剖一种关键生理学的分子基础来解决这些问题。 脊椎动物进化过程中的创新。具体地说，我们将研究一种独特的变构的进化 鳄鱼红细胞血红蛋白(Hb)功能的调节机制。这一独特的模式 变构调节控制有助于鳄鱼屏气潜水的非凡能力。vbl.使用 祖先蛋白质复活与基于位点的组合蛋白质工程方法相结合 定向突变，我们将检查序列突变步骤在小说进化中的影响 鳄鱼HB的变构机制。我们还将深入了解HB变化的结构性基础 功能，因为X射线结晶学实验将揭示原子分辨率的生物物理机制。这个 该项目的具体目标如下：(1)确定导致 新蛋白质功能的进化，并量化它们的相加和非相加效应；以及(2)鉴定和 描述导致功能转变的生物物理机制(新功能的获得、丧失 祖传功能的)。结合起来，实现特定的目标1和2将揭示一种 关键的生理创新，并将提供对这些创新的途径的一般见解 进化。

The step-by-step evolution of novel phenotypes is central to several fundamental questions in biology. In studies of novel protein functions, the problem becomes experimentally tractable if it is possible to identify and functionally characterize the complete set of causative mutations. With such a system, it is possible to address key questions: Do novel functions evolve via the successive fixation of beneficial mutations that each produce an adaptive change in phenotype when they first arise? Alternatively, are evolutionary transitions in protein function facilitated by neutral mutations that produce no adaptive benefit when they first arise, but which potentiate the function-altering effects of subsequent mutations? By reconstructing all possible mutational pathways that connect ancestral and descendant proteins it is also possible to address fundamental questions about the roles of contingency and determinism in protein evolution. For example: Can novel functions evolve from any possible ancestral starting point, or are specific evolutionary outcomes contingent on prior history? We will address these questions by experimentally dissecting the molecular basis of a key physiological innovation during vertebrate evolution. Specifically, we will examine the evolution of a unique allosteric mechanism for regulating hemoglobin (Hb) function in the red blood cells of crocodilians. This unique mode of allosteric regulatory control contributes to crocodilians’ extraordinary capacities for breath-hold diving. Using ancestral protein resurrection in conjunction with a combinatorial protein engineering approach based on site- directed mutagenesis, we will examine the effects of sequential mutational steps in the evolution of the novel allosteric mechanism of crocodilian Hb. We will also obtain insights into the structural basis of the change in Hb function, as X-ray crystallography experiments will reveal biophysical mechanisms at atomic resolution. The specific aims of the project are as follows: (1) Identify the specific mutations that are responsible for the evolution of the novel protein function, and quantify their additive and nonadditive effects; and (2) Identify and characterize the biophysical mechanisms responsible for the functional transition (gain of novel function, loss of ancestral function). In combination, accomplishing Specific Aims 1 and 2 will reveal the molecular basis of a key physiological innovation and will provide general insights into the pathways by which such innovations evolve.