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）中的同种型比其在内质网（微粒体，或Mc b5）中的对应物稳定得多，并且还具有更负的还原电位，这是与电子转移蛋白的功能相关的参数。 从家蝇（HF b5）的孤独的b5表现出中等的稳定性和氧化还原性能。 该项目将建立在过去几年的成功努力，旨在了解不同的生物物理和功能特性的b5亚型的性质，与多肽动力学发挥的关键作用的新的重点。 因此，这些研究的目的是探讨一个假设，即血红素从b5的解离是由高振幅低频合作多肽运动，这是不同的b5亚型从不同的进化谱系。 为此，一个实验方法的基础上自然状态的氢-氘交换NMR光谱监测将结合一个新兴的计算方法（副本交换分子动力学; REX-MD），大大加快了采样率的分子构象相比，以前的方法。 在一个给定的b5亚型的多肽表现出的动态特性最终由其与血红素的相互作用的性质和强度，有必要进行全面的研究，包括全息（血红素结合）和载脂蛋白（血红素自由）状态的蛋白质。 因此，将通过NMR光谱法测定大鼠OM apo-b5的溶液结构，并与大鼠Mc apo-b5的已发表结构进行比较。 以这种方式获得的见解将通过相应的REX-MD模拟进行改进。 第三个目标是通过合理设计大鼠OM b5突变体，其稳定性大于野生型蛋白质，来测试对影响b5稳定性和动力学的因素的不断发展的理解。 除了提供洞察自然的解决方案，以调整b5的性质，其在不同的生物体中的作用，从这个项目的结果持有更广泛的影响，通过（1）提高对稳定的因素，从嗜热生物相对于他们的嗜温对应物的含辅因子的蛋白质的一般理解;以及（2）合理设计具有有用应用的血红素蛋白的相关能力，例如在高温下操作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，既说明概念，也强调科学是如何完成的现实。 这可以是一种有效的手段，刺激本科生的兴趣，在从事研究项目，无论是在PI实验室和他们的同事。 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，在区域和国家会议上介绍他们的结果。

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.