Probing the Impact of Evolutionary Divergence on Structure, Function, Stability and Dynamics of Cytochrome b5

生物信息学的进展揭示了昆虫和植物中编码细胞色素 b5 (b5) 的单个基因，细胞色素 b5 是一种膜锚定电子转移血红素蛋白。 类似基因的复制和随后的进化分歧为哺乳动物提供了两种 b5 亚型，每种亚型在不同的亚细胞细胞器中发挥专门的作用。存在于线粒体外膜 (OM b5) 中的同种型比内质网（微粒体，或 Mc b5）中的同种型更稳定，并且还具有更负的还原电位，这是与电子传递蛋白功能相关的参数。 来自家蝇的单独 b5 (HF b5) 表现出中等稳定性和氧化还原特性。 该项目将建立在过去几年的成功努力的基础上，旨在了解 b5 同工型的不同生物物理和功能特性的本质，并重新强调多肽动力学所发挥的关键作用。 因此，这些研究旨在探讨血红素与 b5 的解离是由高振幅低频协同多肽运动控制的假设，对于来自不同进化谱系的 b5 亚型而言，这种运动是不同的。 为此，基于核磁共振波谱监测的本征氢-氘交换的实验方法将与新兴的计算方法（复制交换分子动力学；REX-MD）相结合，与以前的方法相比，该方法大大加快了分子构象的采样率。 给定 b5 同工型中的多肽所表现出的动态特性最终取决于其与血红素相互作用的性质和强度，因此需要对蛋白质的全息（血红素结合）和 apo（无血红素）状态进行全面研究。 因此，大鼠 OM apo-b5 的溶液结构将通过 NMR 光谱确定，并与已发表的大鼠 Mc apo-b5 的结构进行比较。 以这种方式获得的见解将通过相应的 REX-MD 模拟得到完善。 第三个目标是通过合理设计比野生型蛋白稳定性更高的大鼠 OM b5 突变体，测试对影响 b5 稳定性和动态的因素的理解。 除了深入了解大自然的解决方案以调整 b5 在不同生物体中的作用外，该项目的结果还有望通过以下方式产生更广泛的影响：(1) 提高对稳定嗜热生物体中含辅因子的蛋白质相对于嗜温生物体中的含辅因子蛋白质的因素的一般了解； (2) 合理设计具有有用应用的血红素蛋白的相关能力，例如在高温下运行的生物反应器中的电子载体。 此外，PI 经常在课堂上使用该项目的最新发现，既可以说明概念，又可以强调科学是如何进行的现实。 这可以成为激发本科生对在 PI 实验室及其同事的实验室开展研究项目的兴趣的有效手段。 鼓励所有参与该项目的本科生在内部讨论他们的结果，并在可能的情况下在地区和国家会议上展示他们的结果。

Progress in bioinformatics has revealed a single gene in insects and plants that codes for cytochrome b5 (b5), a membrane-anchored electron transfer heme protein. Duplication of an analogous gene and subsequent evolutionary divergence has provided mammals with two b5 isoforms, each playing specialized roles in distinct sub-cellular organelles. The isoform that resides in the outer mitochondrial membrane (OM b5) is considerably more stable than its counterpart in the endoplasmic reticulum (microsomal, or Mc b5), and also has a more negative reduction potential, a parameter related to function in electron transfer proteins. The lone b5 from house fly (HF b5) exhibits intermediate stability and redox properties. This project will build upon successful efforts over the past several years, aimed at understanding the nature of the divergent biophysical and functional properties of b5 isoforms, with a new emphasis on the critical role played by polypeptide dynamics. Hence, these studies are aimed at probing a hypothesis that dissociation of heme from b5 is governed by high amplitude low-frequency cooperative polypeptide motions, which differ for b5 isoforms from different evolutionary lineages. To this end, an experimental approach based on native-state hydrogen-deuterium exchange monitored by NMR spectroscopy will be combined with an emerging computational methodology (replica exchange molecular dynamics; REX-MD) that greatly accelerates the sampling rate of molecular conformations in comparison to previous methods. The dynamic properties exhibited by the polypeptide in a given b5 isoform are ultimately governed by the nature and strength of its interactions with the heme, necessitating that a comprehensive study encompass both the holo (heme-bound) and apo (heme-free) states of the protein. Consequently, the solution structure of rat OM apo-b5 will be determined by NMR spectroscopy and compared to the published structure of rat Mc apo-b5. Insights gained in this manner will be refined through corresponding REX-MD simulations. The third objective is to test the developing understanding of factors affecting stability and dynamics in b5 through the rational design of rat OM b5 mutants with stability greater than that of the wild-type protein. In addition to providing insight into nature's solutions to tuning the properties of b5 for its roles in different organisms, results from this project hold promise of a broader impact by (1) improving general understanding of the factors that stabilize cofactor-containing proteins from thermophilic organisms relative to those of their mesophilic counterparts; and (2) the related ability to rationally design heme proteins with useful applications, such as electron carriers in bioreactors operating at elevated temperatures. Further, the PIs routinely use recent findings from the project in the classroom, both to illustrate concepts and to highlight the reality of how science is done. This can be an effective means of stimulating undergraduate student interest in undertaking research projects, both in the PIs laboratories and in those of their colleagues. All undergraduates participating in the project are encouraged to discuss their results in house and, when possible, to present their results at regional and national meetings.