Molecular structural studies of protein complexes for biotechnology and metabolic engineering

我的研究项目使用分子结构方法来理解和设计对生物技术，特别是代谢工程非常重要的关键酶和结合蛋白。项目1旨在设计具有独特有用的氧化还原可切换结合特性的链霉亲和素的新形式。我们最近取得了突破，发现了在链霉亲和素的关键环中引入二硫键是如何产生氧化还原依赖的生物素结合的。用温和的还原剂打破其中一个二硫键，使生物素的解离速度增加一千倍以上，首次允许在生理条件下可逆转地形成和破坏紧密结合的生物素络合物。这种突变蛋白对某些应用很有用，但对于需要快速解离动力学的应用来说，结合得太紧了。为了解决这一缺点，我们提出了一种基于结构的策略来设计和表征一套下一代突变体，在还原状态下具有更快的解离动力学，而在氧化状态下保持极慢的解离动力学。这些突变体将在生物技术中有许多应用，包括在本提案的项目2和3中立即应用于发现蛋白质相互作用伙伴。项目2探索植物中磷酸蛋白磷酸酶(PPP)家族新成员的结构-功能关系。通过解决AtRLPH2的结构和表征不寻常的底物特异性，我们最近发现了一种识别双重磷酸化底物的新机制，证实了RLPH2参与MAP激酶级联调控的磷酸蛋白质组数据。使用项目1中的工具，我建议进一步探索AtRLPH2如何调节MAP激酶，以及确定其他非规范植物蛋白磷酸酶的基本结构-功能关系，如AtSLP1。项目3进一步发展了对生物合成苄基异喹啉生物碱(BIA)化合物的关键酶的研究。我们对N-甲基转移酶(NMTs)的基础结构-功能研究已经建立了特异性和催化的关键分子机制。这些发现表明如何设计具有新的特异性的NMT突变体，为代谢工程创造新的工具。我们还进行了BIA O-甲基转移酶的结构和功能的研究，以及将茶碱转化为可待因和吗啡的途径的末端步骤的酶。我们最近对可待酮还原酶的结构和功能的研究表明，新的蛋白质-蛋白质相互作用可能对次生代谢物的生物合成具有更广泛的意义。项目1中的工具将帮助我们探索其中一些蛋白质-蛋白质相互作用。我的研究计划关注的是在加拿大快速发展的生物技术行业中既有短期应用又有长期应用的问题，这些问题将促进生物学基础知识的发展。

My research program uses a molecular structural approach to understand and engineer key enzymes and binding proteins important for biotechnology, especially metabolic engineering. Project 1 aims to engineer novel forms of streptavidin with uniquely useful redox-switchable binding properties. We recently made a breakthrough, discovering how the introduction of disulphide bonds into a key loop in streptavidin creates redox-dependent binding to biotin. Breaking one of these disulphide bonds with a mild reducing agent increases the rate of dissociation for biotin by over a thousand-fold, allowing for the first time the reversible formation and breakdown of tight-binding biotin complexes under physiological conditions. This mutein is useful for certain applications but binds too tightly for applications where fast dissociation kinetics are required. To address this shortcoming, we propose a structure-based strategy to design and characterize a suite of next-generation muteins with faster dissociation kinetics in the reduced state while retaining extremely slow dissociation kinetics in the oxidized state. These muteins would have many applications in biotechnology, including immediate applications to discover protein interaction partners in projects 2 and 3 of this proposal. Project 2 explores structure-function relationships in novel members of the phosphoprotein phosphatase (PPP) family in plants. By solving the structure and characterizing the unusual substrate specificity of AtRLPH2, we recently discovered a novel mechanism to recognize dually phosphorylated substrates, corroborating phosphoproteomic data implicating RLPH2 in the regulation of MAP kinase cascades. Using tools from project 1, I propose to further explore how AtRLPH2 regulates MAP kinases, as well as to determine foundational structure-function relationships for other noncanonical plant protein phosphatases, such as AtSLP1. Project 3 further develops studies on enzymes central to the biosynthesis of benzylisoquinoline alkaloid (BIA) compounds. Our foundational structure-function studies of N-methyltransferasse (NMTs) have established key molecular mechanisms underlying specificity and catalysis. These findings indicate how to design NMT muteins with novel specificities, creating new tools for metabolic engineering. We have also undertaken structure-function studies of BIA O-methyltransferases, as well as the enzymes in the terminal steps of the pathway converting thebaine to codeine and morphine. Our recent structure-function studies of codeinone reductase suggest the possible importance of novel protein-protein interactions with wider implications on the biosynthesis of secondary metabolites. Tools from project 1 will help us explore some of these protein-protein interactions. My research program focuses on problems that will have both short-term applications in Canada's rapidly growing biotechnology industry, as well as long-term applications advancing basic knowledge in biology.